Anticancer therapeutic intervention

ABSTRACT

The present invention is directed to a method of treating cancer using interfering RNA duplexes to mediate gene silencing. The present invention is also directed to interfering RNA duplexes and vectors encoding such interfering RNA duplexes.

FIELD OF THE INVENTION

The present invention is directed to a method of treating cancer usinginterfering RNA duplexes to mediate gene silencing. The presentinvention is also directed to interfering RNA duplexes and vectorsencoding such interfering RNA duplexes.

DESCRIPTION OF RELATED ART

Regardless of their origin, tumours require a constant supply ofnutrients to support their characteristic unabated growth. In fact,tumour cells may consume more nutrients than required for their ownmetabolic needs (Medina et al., 1992b), and exhibit distinct metabolicprofiles compared to their normal cellular counterparts. Tumours aresupplied with nutrients to fuel their metabolic needs through thecollective processes of angiogenesis and the prodigious expression ofnutrient transporters in the plasma membranes of constituent cells.Amino acids are the primary source of cellular nitrogen, used fornucleotide, glutathione, amino sugar and protein synthesis. Thevoracious and apparently wasteful amino acid metabolism of malignanttumours leads to, among other things, negative nitrogen balance in thehost with cancer. Moreover, the carbon skeletons of amino acids areoften utilized as an oxidative fuel source for ATP generation inaddition to glucose and fatty acids, and may also contribute to steroland lipid biosynthesis (Baggetto, 1992; Medina et al., 1992a). Comparedto normal cells or tissues, cancer cells display enhanced and alteredchannelling of amino acids into select metabolic pathways, often inconcert with the aerobic glycolysis characteristic of tumours (Baggetto,1992) and (Mazurek et al., 2003). Solid tumours are often poorlyvascularized, especially in the nascent avascular phase characteristicof neoplasia and metastases, so they must have efficient mechanisms forextracting plasma amino acids in order to compete with host tissues(Medina et al., 1992b). Cancer, in turn, is a microcosm of evolution,with the “fittest” cells enduring through adaptation to the localmicroenvironment; as a result, amino acid transporters with propertiesthat impart growth and survival advantages are selected for andexpressed, often at augmented levels compared to the parent tissue.

Prior to the advent of mammalian amino acid transporter cloning andisolation, Christensen proposed that specific amino acid transporterscould be upregulated in transformed cells to support the high levels ofprotein synthesis necessary for growth and proliferation (Christensen,1990). A search of the human expressed sequence tag (EST) database atthe Cancer Genome Anatomy Project (CGAP) website(http://cgap.nci.nih.gov), using the “cDNA Virtual northern” tool todetermine the expression levels of the classic neutral amino acidtransport “systems” A, ASC, L and N, in normal and cancerous tissuesrevealed that while five transporters are significantly enhanced overallin cancerous tissues, two stand out—LAT1 (large neutral amino acidtransporter 1) and ASCT2 (ASC amino acid transporter 2) (Fuchs et al.,2005). ASCT2 and LAT1 are both upregulated three-fold (collectively) ina variety of cancerous tissues where their expression pattern is almostidentical (Fuchs et al., 2005).

LAT1 has long been associated with cancerous or proliferative cells.When full-length LAT1 was first isolated and characterized in 1998, itwas shown not to be expressed in rat liver but was detected in rathepatoma (dRLh-84) and hepatocarcinoma (FAA-HTC1) cell lines (Kanai etal., 1998), which is consistent with the expression pattern of ASCT2 inhuman liver as discussed below. Northern blot analysis with human celllines revealed TA1 expression in the choriocarcinoma cell line JEG-3 andbreast carcinoma cell line MDA-A1 (Sang et al., 1995), as well as instomach signet ring cell carcinoma (KATOIII), lung small cell carcinoma(RERF-LC-MA) and malignant melanoma (G-361) (Kanai et al., 1998). Both4F2hc and LAT1 were detected in several leukemia cell lines. RERF-LC-MAlung small cell carcinoma cells, HeLa uterine cervical carcinoma cellsand T-24 bladder carcinoma cells (Yanagida et al., 2001). The resultswith the T-24 cells have been confirmed by confocal immunofluorescencemicroscopy, which revealed colocalization of LAT1 and 4F2hc in theplasma membrane (Kim et al., 2002). LAT1 and 4F2hc have also been shownto colocalize in the plasma membrane of KB human oral epidermoidcarcinoma cells (Yoon et al., 2005), and may be important for oralsquamous epithelial carcinogenesis, as immunohistochemical staining hasshown that their expression increases during progression of oral normalmucosa to oral squamous cell carcinoma (Kim et al., 2004b).

Kühne et al., (Kuhne et al., 2007) discloses genetic polymorphisms ofthe LAT1 and LAT2 genes and relates such polymorphisms to thepharmacokinetics of melphalan. Shennan et al., (Shennan et al., 2008)discloses that inhibiting LAT1 reduces the growth of human breast cancercells. Nawashiro et al., (Nawashiro et al., 2006) discloses that LAT1 isa potential molecular target in human astrocytic tumors. Yamauchi etal., (Yamauchi et al., 2009) and Kim et al., (Kim et al., 2008) disclosethe use of LAT1 inhibitors to block tumor activity of various cancercells. In the context of the requirement of the 4F2hc chaperone forenabling LAT1 activity, real-time quantitative RT-PCR revealed similarlevels of LAT1 and 4F2hc mRNA in KB cells (Yoon et al., 2005). Thisconflicts with the data from T-24 cells where LAT1 levels were ˜1.5 foldhigher than 4F2hc (Kim et al., 2002) and with results in MDA-MB-231 andMCF-7 human breast cancer cells, where LAT1 mRNA was ˜4.3- and ˜4.9-foldhigher than 4F2hc (Shennan et al., 2004). Based on the data by (Fuchs etal., 2005), it appears that the overall abundance of 4F2hc mRNA is onpar with that of LAT1, but it should be kept in mind that protein andmRNA levels are not always linearly correlated due to the multiplemechanisms of gene expression control. Moreover, cells undoubtedlypossess mechanisms that adequately match protein levels of bothcomponents, such that 4F2hc is not rate-limiting for vital LAT1-relatedfunctions.

When colon cancer RCN-9 cells were injected into the spleen of rats, thesize of the resultant metastatic liver tumors was directly correlated toLAT1 expression (Ohkame et al., 2001; Tamai et al., 2001). Thus, it hasbeen proposed that inhibiting LAT1 function could serve as a potentialtherapeutic for many types of cancers (Kanai et al., 2001). To date,studies targeting LAT1 specifically are scarce. However, in vitro LAT1antisense expression in non-human hepatic tumor cells resulted in amodest though statistically significant decrease in cell number,viability and S-phase cells over a 5-day period relative to controlsdespite the absence of a significant decrease in L-type transport overthis period (Storey et al., 2005).

Antisense oligonucleotides directed against 4F2hc have been shown toinhibit Na⁺-independent isoleucine transport in C6-BU-1 rat glioma cells(Broer et al., 1997), and leucine uptake in BeWo human cytotrophoblastcells (Kudo et al., 2004), but the effects on cell growth were notreported. An anti-4F2hc antibody inhibited the proliferation of avariety of tumor cell lines (Yagita et al., 1986), but it is possiblethat these effects were mediated through the collective effects on other4F2 “light chains” in addition to LAT1, or to non-transport relatedfunctions of 4F2hc (Feral et al., 2005). Knockdown of LAT1 in T-24 cellswith a LAT1-siRNA against nucleotides 1173-1194 in vitro led to adecrease in L-CSNO uptake (U et al., 2005), but it is not reportedwhether cell survival was affected. Thus, mechanistic studies linkingthe LAT1 “light chain” to cancer growth remain to be reported.

The use of siRNA to inhibit LAT1 gene expression have been disclose byseveral groups (Kim et al., 2006a; Kim et al., 2006b; Kaneko et al.,2007; Pinho et al., 2007a; Yin et al., 2008; Kuhne et al., 2009; Nicklinet al., 2009; Xia et al., 2010; Liang et al., 2011; Miko et al., 2011;Ohkawa et al., 2011; Denoyer et al., 2012; Hayashi et al., 2012; Dickenset al., 2013; Hayashi et al., 2013; Youland et al., 2013; Gaccioll etal., 2015; Habermeer et al., 2015; Wongthal et al., 2015; Furugen etal., 2016; Polet et al., 2016; Straka at al., 2016; Tomblin et al.,2016; Wei at al., 2016; Xu et al., 2016; Liu et al., 2017).

More recently, LAT1 has been suggested as a marker of cancer prognosisin the types of cancer listed in the following table:

Kidney Renal cell carcinoma (Betsunoh et al., 2013) Urothelialcarcinomas (Eltz et al., 2008) Cell carcinoma of the upper urinary(Nakanishi et al., 2007) tract Lung Non-small cell lung cancer (Kaira etal., 2008b; Imai et al., 2009; Kaira et al., 2009c; Kaira et al., 2009d;Kaira et al., 2010b; Kaira et al., 2010a; Kyoichi, 2010; Takeuchi etal., 2010; Kaira et al., 2011b; Kaira et al., 2012a; Chuntao et al.,2015) Pulmonary adenocarcinoma (Kaira et al., 2010b) Squamous cellcarcinoma of the lung (Kaira et al., 2009b) Lung cancer (Kaira et al.,2007; Kaira et al., 2011a) Neuroendocrine tumors of the lung (Kaira etal., 2008c; Kyoichi et al., 2008) Malignant pleural mesothelioma (Kairaet al., 2011c) Colon Rectal cancer (Ebara et al., 2010) Breast BreastCancer (Furuya et al., 2012; Emer et al., 2013; Fukumoto et al., 2013)Pancreas Pancreatic cancer (Kaira et al., 2012b; Yanagisawa et al.,2012; Kaira et al., 2015a) Gastrintestinal Gastric carcinoma (Ichinoe etal., 2011; Ichinoe et al., 2015) Oesophageal cancer (Suzuki et al.,2014) Blood Multiple myeloma (Isoda et al., 2014) Thymic carcinomas(Kaira et al., 2009a) Hepatobilbiar Biliary tract cancer (Kaira et al.,2014; Yanagisawa et al., 2014) Hepatocellular carcinoma (Li et al.,2013; Masashi et al., 2014) Adenoid cystic carcinoma (Kaira et al.,2013) Metastatic Primary metastatsis (Kaira et al., 2008a) LiverMetastasis (Kaoru et al., 2011) Brain Glioma (Keyaerts et al., 2007;Stockhammer et al., 2008; Okubo et al., 2010) Astrocytic tumors(Nawashiro et al., 2006) Head & Neck Oral squamous cell carcinoma (Kimet al., 2004a; Nobusawa et al., 2013) Tongue cancer (Toyoda et al.,2014a) Hypopharyngeal squamous cell (Toyoda et al., 2014b) carcinomaProstate Prostate cancer (Sakata et al., 2009; Wang et al., 2011; Segawaet al., 2013; Yanagisawa et al., 2015) Uterus Endometrioidadenocarcinoma (Watanabe et al., 2014)

System ASC transport activity is ubiquitous and characterized by itspreference for small neutral amino acids including alanine, serine, andcysteine. The system ASC of neutral amino acid transporters (SLC1A4 andSLC1A5) belongs to the solute carrier family-1 (SLC1), which alsoincludes the high-affinity glutamate transporters. Human ATB0 wasidentified by RT-PCR and enzymatic restriction analysis in the humanproximal tubule cell line HKPT and corresponds to rodent ASCT2. The twoASC transporters exhibit distinct substrate selectivity. SLC1A4 encodesthe sodium-dependent amino acid transporter ASCT1, which acceptsL-alanine, Lserine, L-theonine, and L-cysteine in a stereospecificmanner. ASCT2, the second isoform of the ASC transport system, isencoded by SLC1A5. In the kidney and intestine, ASCT2 is present in thebrush-border membranes of the proximal tubule cells and enterocytes,respectively. In addition to the typical system ASC substrates, it alsoaccepts L-glutamine and L-asparagine at higher affinity as well asmethlonine, leucine, and glycine with lower affinity. Both ASCT1 andASCT2 mediate the sodium-dependent obligatory exchange of substrateamino acids (Pinho et al., 2007b).

More recently, ASCT2 has been suggested as a marker of cancer prognosisin the types of cancer listed in the following table:

Breast Breast Cancer (Betsunoh et al., 2013; Kim et al., 2013) BrainNeuroblastoma (Ren et al., 2015) Colon Rectal cancer (Witte et al.,2002) Gastrintestinal Oesophageal cancer (Honjo et al., 2016) Head &Neck Lacrimal gland adenocarcinoma (Koo et al., 2015) Tongue cancer(Toyoda et al., 2014a) Laryngeal squamous cell carcinoma (Nikkuni etal., 2015) Thyroid medullary carcinoma (Kim et al., 2016) HepatobilbiarHepatocellular carcinoma (Ge et al., 2015) Kidney Renal cell carcinoma(Liu et al., 2015) Lung Non-small cell lung cancer (Shimizu et al.,2014) Metastatic Primary metastatsis (Kim et al., 2014) PancreasPancreatic cancer (Kaira et al., 2015a; Kaira et al., 2015b) SkinMelanoma (Wang et al., 2014)

ASCT2 is expressed in colorectal adenocarcinomas and patient survivaldecreased with increased percentage of ASCT2-positive cancer cells.These results indicate that ASCT2 is expressed in a significant numberof colorectal adenocarcinomas, and that ASCT2 expression is associatedwith aggressive biological behavior (Witte et al., 2002). It has beenproposed that ASCT2 appears to be required for the glutamine metabolismin both nonmalignant and malignant prostate. However, ASCT2-positiveprostate adenocarcinoma seems to be related to a more aggressivebiological behavior. ASCT 2 seems to be involved in tumor progression(Li et al., 2003; Wang et al., 2015). ASCT2 expression has a crucialrole in the metastasis of pulmonary adecocarccinomas, and is a potentialmolecular marker for predicting poor prognosis after surgery (Shimizu etal., 2014). ASCT2 expression was also found to play an important role intumour cel growth, and is a promising pathological marker for predictinga worse outcome in pancreatic cancer (Kaira et al., 2015b; Kyoichi etal., 2015). High ASCT2 expression was also found to be significantlyassociated with poor prognosis and survival of neuroblastoma patients(Ren et al., 2015). In addition, others have suggested that ASCT2suppression exerts proapoptotic effects transcending those of glutaminestarvation alone (Bryan et al., 2004; Fuchs et al., 2004). Theimportance of ASCT2 expression in melanoma was confirmed by shRNAknockdown, which inhibited glutamine uptake, mTORC1 signalling and cellproliferation (Wang et al., 2014).

The use of siRNA to inhibit ASCT2 gene expression have been disclose byseveral groups (Wieland et al., 2005; Nicklin et al., 2009; Okudaira etal., 2011; Barel et al., 2012; Hassanein et al., 2013; Hassanein et al.,2015; Corbet et al., 2016; Straka et al., 2016; Zhou et al., 2016; Leeet al., 2017).

RNA interference (“RNAi”) is a recently discovered mechanism ofpost-transcriptional gene silencing in which double-stranded RNAcorresponding to a gene (or coding region) of interest is introducedinto an organism, resulting in degradation of the corresponding mRNA.The phenomenon was originally discovered in Caenorhabditis elegans (Fireet al., 1998).

Unlike antisense technology, the RNAi phenomenon persists for multiplecell divisions before gene expression is regained. The process occurs inat least two steps: an endogenous ribonuclease cleaves the longer dsRNAinto shorter, 21- 22- or 23-nucleotide-long RNAs, termed “smallinterfering RNAs” or siRNAs (Hannon, 2002). The siRNA segments thenmediate the degradation of the target mRNA. RNAi has been used for genefunction determination in a manner similar to but more efficient thanantisense oligonucleotides. By making targeted knockouts at the RNAlevel by RNAi, rather than at the DNA level using conventional geneknockout technology, a vast number of genes can be assayed quickly andefficiently. RNAi is therefore an extremely powerful, simple method forassaying gene function.

RNAi has been shown to be effective in cultured mammalian cells. In mostmethods described to date, RNAi is carried out by introducingdouble-stranded RNA Into cels by microinjection or by soaking culturedcells in a solution of double-stranded RNA, as well as transfecting thecells with a plasmid carrying a hairpin-structured siRNA expressingcassette under the control of suitable promoters, such as the U6. H1 orcytomegalovirus (“CMV”) promoter (Elbashir et al., 2001; Harborth etal., 2001; Lee et al., 2001; Brummelkamp et al., 2002; Miyagishi et al.,2002; Paddison et al., 2002; Paul et al., 2002; Sui et al., 2002; Xia etal., 2002; Yu et al., 2002). The gene-specific inhibition of geneexpression by double-stranded ribonucleic acid is generally described inU.S. Pat. No. 6,506,559, which is incorporated herein by reference.Exemplary use of siRNA technology is further described in Published U.S.Patent Application No. 2003/01090635 and Published U.S. PatentApplication No. 20040248174, which are incorporated herein by reference.Davis (Davis, 2009) describes the targeted delivery of siRNA to humansusing nanoparticle technology.

SUMMARY OF THE INVENTION

An object of the present invention is to use an RNA interferencetechnique to down regulate the expression of the LAT1 and/or ASCT2 genesin order to treat or prevent cancer. Preferred cancers that can betreated or prevented by the present invention include bladder, brain,colon, head and neck, kidney, liver, lung, lymph node, mammary gland,muscle, ovary, pancreas, skin and stomach cancers. The compositions (ormolecules) of the invention comprises or consists of short interferingnucleic acid molecules (siNA) and related compounds including, but notlimited to, siRNA. The present invention encompasses compositions andmethods of use of siNA including, but not limited to short interferingRNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), antagomirsand short hairpin RNA (shRNA) capable of mediating RNA interference. Inone embodiment, the siNA molecule of the invention can be incorporatedinto RISC (RNA-induced silencing complex).

A further object of the present invention is to provide a siRNA moleculethat efficiently down regulates the expression of the LAT1 gene and/orASCT2 gene.

Accordingly, in a first aspect, the invention relates to a siNAmolecule, wherein said molecule specifically targets at least onesequence selected from SEQ ID NO: 1-SEQ ID NO: 104 or a variant thereofand SEQ ID NO: 209-SEQ ID NO: 297 or a variant thereof. In analternative embodiment, the invention relates to an siNA moleculewherein said molecule specifically targets at least one sequencecomplementary to at least one sequence selected from SEQ ID NO: 1-SEQ IDNO: 104 or a variant thereof and SEQ ID NO: 209-SEQ ID NO: 297 or avariant thereof. In one embodiment, the invention relates to an isolatedsiNA molecule, preferably an isolated siRNA molecule.

In one embodiment, the siNA molecule reduces expression of the(preferably, human) LAT 1 and/or ASCT2 gene when introduced into a cell.

In one embodiment, the siNA molecule specifically targets at least onesequence selected from SEQ ID No 4, 6, 10, 13, 22, 34, 58, 61, 81, 83,87 and 95 to 104 or a variant thereof. Preferably, the siNA moleculetargets a sequence selected from SEQ ID NO 6, 22, 34, 58 and 61 or avariant thereof. Even more preferably, the siNA targets SEQ ID NO: 61 or58 to a variant thereof. Preferably, the siNA molecule reducesexpression of the LAT1 gene when expressed into a cell.

In an alternative embodiment, the siNA molecule specifically targets atleast one sequence selected from SEQ ID NO: 209, 216, 225, 226, 228, 235to 238, 245, 260, 264, 267, 271, 272, 278, 279 and 281 to 297.Preferably, the siNA molecule targets a sequence selected from SEQ ID NO225, 237, 267 or 278 or a variant thereof. Even more preferably, thesiNA targets SEQ ID NO: 267 or 278 or a variant thereof. Preferably thesiNA molecule reduces expression of the ASCT2 gene when expressed into acell.

In a further embodiment, the siNA preferably comprises a double-strandedRNA molecule, whose antisense strand is substantially complementary toany of SEQ ID NO: 1-SEQ ID NO: 104, more preferably SEQ ID No 4, 6, 10,13, 22, 34, 58, 61, 81, 83, 87 and 95 to 104, even more preferably SEQID NO 6, 22, 34, 58 and 61, and most preferably SEQ ID NO: 61 or 58 orany variant thereof, and its sense strand will comprise an RNA sequencecomplementary to the sense strand, wherein both strands are hybridisedby standard base pairing between nucleotides. In a further embodiment,said sense stand comprises or consists of a sequence selected from SEQID NO: 105 to 208, preferably SEQ ID NO: 108, 110, 114, 117, 126, 138,162, 165, 185, 187, 191 and 199 to 208, more preferably SEQ ID NO: 110,126, 138, 162 and 165 and most preferably SEQ ID NO: 162 or 165 or avariant thereof. The corresponding antisense strands are described inFIG. 26. Preferably, the siNA molecule reduces expression of the LAT1gene when expressed into a cell.

In an alternative embodiment, the siNA preferably comprises adouble-stranded RNA molecule, whose antisense strand is substantiallycomplementary to any of SEQ ID NO: 209-SEQ ID NO: 297, more preferablySEQ ID NO: 209, 216, 225, 226, 228, 235 to 238, 245, 260, 264, 267, 271,272, 278, 279 and 281 to 297, even more preferably SEQ ID NO 225, 237,267 or 278 and most preferably SEQ ID NO: 267 or 278 or any variantthereof, and its sense strand will comprise an RNA sequencecomplementary to the sense strand, wherein both strands are hybridisedby standard base pairing between nucleotides. In a further embodiment,said sense stand comprises or consists of a sequence selected from SEQID NO: 298 to 386, preferably, SEQ ID NO: 298, 305, 314, 315, 317,324-327, 334, 349, 353, 356, 360, 361, 367, 368 and 370 to 386, morepreferably SEQ ID NO: 314, 326, 36 and 367 or a variant thereof, andeven more preferably SEQ ID NO: 356 or 367 or any variant thereof. Thecorresponding antisense strands are described in FIG. 27. Preferably,the siNA molecule reduces expression of the ASCT2 gene when expressedinto a cell.

Within the meaning of the present invention “substantiallycomplementary” to a target mRNA sequence, may also be understood as“substantially identical” to said target sequence. “Identity” as isknown by one of ordinary skill in the art, is the degree of sequencerelatedness between nucleotide sequences as determined by matching theorder and identity of nucleotides between sequences. In one embodimentthe antisense strand of an siRNA having 80%, and between 80% up to 100%complementarity, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97% or 99% complementarity, to the target mRNAsequence are considered substantially complementary and may be used inthe present invention. The percentage of complementarity describes thepercentage of contiguous nucleotides in a first nucleic acid moleculethat can base pair in the Watson-Crick sense with a set of contiguousnucleotides in a second nucleic acid molecule.

A gene is “targeted” by a siNA according to the present invention when,for example, the siNA molecule selectively decreases or inhibits theexpression of the gene. The phrase “selectively decrease or inhibit” asused herein encompasses siNAs that affect expression of LAT1 and/orASCT2. Alternatively, a siNA targets a gene when the siNA hybridizesunder stringent conditions to the gene transcript, i.e. its mRNA.Capable of hybridizing “under stringent conditions” means annealing tothe target mRNA region, under standard conditions, e.g., hightemperature and/or low salt content which tend to disfavorhybridization. A suitable protocol (involving 0.1×SSC, 68° C. for 2hours) is described in Maniatis, T., et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, 1982, at pages387-389.

Nucleic acid sequences cited herein are written in a 5′ to 3′ directionunless indicated otherwise. The term “nucleic acid” refers to either DNAor RNA or a modified form thereof comprising the purine or pyrimidinebases present in DNA (adenine “A”, cytosine “C”, guanine “G”, thymine“T”) or in RNA (adenine “A”, cytosine “C”, guanine “G”, uracil “U”).Interfering RNAs provided herein may comprise “T” bases, for example at3′ ends, even though “T” bases do not naturally occur in RNA. In somecases these bases may appear as “dT” to differentiatedeoxyribonucleoties present in a chain of ribonucleotides.

In one embodiment of the invention, the siNA molecule is 40 base pairsor fewer in length. Preferably, the siNA molecule is 19 to 25 base pairsin length. In one embodiment, the siNA comprises or consists of a 21nucleotide double-stranded region. Preferably, the siNA has a sense andan anti-sense strand. In an alternative embodiment, the siNA moleculecomprises or consists of a 19 nucleotide double-stranded region. In oneembodiment, the siNA has blunt ends. In an alternative embodiment, thesiNA has 5′ and/or 3′ overhangs. Preferably the overhangs are between 1to 5 nucleotides, more preferably, 2 nucleotide overhangs. The overhangsmay be ribonucleic acids, or deoxyribonucleic acids.

In one embodiment, the siNA molecule according to the inventioncomprises a chemical modification. Preferably, the chemical modificationis on the sense strand, the antisense strand or both. Examples ofchemical modifications include phosphorothioate internucleotidelinkages, 2′-OMethylation, 2′-deoxy-fluoro ribonucleotkides, 2′-deoxyribonucleotides, 5-C methyl nucleotides, inverted deoxybasic residueincorporation or a substitution of uracyl ribose nucleotides withdeoxythymidine nucleotides or combinations thereof.

In one embodiment, the 5′ or 3′ overhangs are dinucleotides, preferablythymidine dinucleotide. In a preferred embodiment, the 5′ or 3′overhangs are deoxythymidines. In one embodiment, the sense strandcomprises at least one, preferably two 3′ overhangs. Preferably, saidsense strand comprises at least one, preferably two 3′ deoxythymidines.In an alternative embodiment, the antisense strand comprises at leastone, preferably two 3′ overhangs. Preferably, said sense strandcomprises at least one, preferably two 3′ deoxythymidines. In a furtherpreferred embodiment, both the sense and antisense strands comprise 3′overhangs as described herein.

In a further embodiment, the siNA molecule preferably comprises adouble-stranded RNA molecule, wherein preferably the sense strand andthe anti-sense strand are selected from the below or a variant thereof.

Anti- Sense sense strand strand 387 388. This sequence is also referredto herein as SEQ ID NO: 58t. 389 390 This sequence is also referred toherein as SEQ ID NO: 58s1 391 392 This sequence is also referred toherein as SEQ ID NO: 58s2 393 394 This sequence is also referred toherein as SEQ ID NO: 61t 395 396 This sequence is also referred toherein as SEQ ID NO: 61s1 397 398 This sequence is also referred toherein as SEQ ID NO: 61s2 399 400 This sequence is also referred toherein as SEQ ID NO: 267t 401 402 This sequence is also referred toherein as SEQ ID NO: 267s1 403 404 This sequence is also referred toherein as SEQ ID NO: 267s2 405 406 This sequence is also referred toherein as SEQ ID NO: 278t 407 408 This sequence is also referred toherein as SEQ ID NO: 278s1 409 410 This sequence is also referred toherein as SEQ ID NO: 278s2

By “variant” as used herein is meant a sequence with 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 83%, 84%,85%, 86%, 87%, 88% %, 9087%, 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, orat least 99% overall sequence identify to the non-variant nucleic orribonucleic acid sequence.

By “down-regulating” is meant a decrease in the expression of LAT1and/or ASCT2 by up to or more than 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%50%, 55% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% when compared to thelevel in a control. Alternatively, the siNA molecule described hereinmay abolish LAT1 and/or ASCT2 expression. The term “abolish” means thatno expression of LAT1 and/or ASCT2 is detectable or that no functionalLAT1 and/or ASCT2 protein is produced. For example, a reduction in theexpression and/or protein levels of at least LAT1 and/or ASCT2expression may be a measure of protein and/or nucleic acid levels andcan be measured by any technique known to the skilled person, such as,but not limited to, any form of gel electrophoresis or chromatography(e.g. HPLC).

Notably, in some embodiments, the siNA molecule (either the 5′ or 3′strand or both) may begin with at least one, preferably two alaninenucleotides. Alternatively, if the target sequence starts with one ortwo alanine sequences, these may not be included (targeted) in the siNAmolecule.

In one embodiment, the target sequence may be characterised by at leastone, preferably two alanine nucleotides at the 3′ end of the sequence,and/or the target sequence lacks at least one, preferably two alaninenucleotides at the 5′ end of the sequence, and/or the target sequencelacks two consecutive alanine nucleotides within the sequence. In apreferred embodiment, the siNA molecules of the invention arecharacterised in that they target sequences with the above properties.

In one embodiment a plurality of species of siNA molecule are used,wherein said plurality of siNA molecules are targeted to the same or adifferent mRNA species.

In one embodiment, the siNA is selected from dsRNA, siRNA or shRNA.Preferably, the siNA is siRNA.

In a further embodiment, the invention relates to a siNA molecule, asherein described for use as a medicament. In one embodiment, theinvention relates to a siNA for use in the treatment of a disordercharacterised by increased expression levels (compared to the levels ina healthy subject) of LAT1 and/or ASCT2.

In another aspect of the invention, there is provided a siNA molecule,as described herein for use in the treatment of cancer.

In a further aspect, the invention relates to the use of at least onesiNA molecule, as described herein in the preparation of a medicamentfor the treatment of cancer.

In another aspect, the invention relates to a method for the treatmentof cancer, the method comprising administering at least one siNAmolecule, as described herein, to a patient or subject in need thereof.

In one embodiment, the cancer is selected from bladder, blood, brain,colon, head and neck, kidney, liver, lung, lymph node, mammary gland,muscle, ovary, pancreas, prostate, skin, stomach and uterus cancer.

In another aspect of the invention there is provided a pharmaceuticalcomposition comprising at least one siNA molecule as described hereinand a pharmaceutically acceptable carrier.

In a further aspect of the invention there is provided a method,preferably an in vitro method of inhibiting amino acid uptake into acell, the method comprising administering a siNA as defined herein to acell. Preferably, the amino acid uptake is sodium-independent leucineuptake. Alternatively, the amino acid uptake is sodium-dependent alanineuptake. In one embodiment, amino acid uptake is inhibited by up to ormore than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared tothe level in a control.

In a further aspect of the invention, there is provided a method ofdecreasing cell proliferation, the method comprising administering atleast on siNA as described herein to a cell. In one embodiment, saiddecrease in cell proliferation may be up to or more than 10%, 20%, 30%,40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a control.

In a yet further aspect of the invention, there is provided a method ofreducing tumour volume, preferably in a patient, the method comprisingadministering at least one siNA as described herein. In one embodiment,said decrease in tumour volume may be up to or more than 10%, 20%, 30%,40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a control.

In on embodiment, of the above-described methods at least one LAT-1 siNAor at least one ASCT2 siNA is administered. In an alternativeembodiment, at least one LAT-1 siNA and at least one ASCT2 siNA isadministered

In another embodiment, the invention relates to methods of reducingcancer cell proliferation comprising treating the cells with an siNA ofthe invention in combination with one or more anti-cancer agents knownin the art, preferably wherein the anti-cancer agent comprises ananti-antineoplastic agent, more preferably a cytotoxic antineoplasticagent and most preferably 5-fluoruracil (5-FU), cisplatin (Cisp) and/oroxaiplatin (Oxa).

The invention also relates to methods of treating cancer comprisingadministrating an siNA of the invention in combination with one or moreanti-cancer agents known in the art, preferably to a patient in needthereof, preferably wherein the anti-cancer agent comprises ananti-antineoplastic agent, more preferably a cytotoxic antineoplasticagent and most preferably 5-fluoruracil (5-FU), cisplatin (Cisp) and/oroxaliplatin (Oxa). The invention further relates to pharmaceuticalcompositions comprising the siNA of the invention and the one or moreanti-cancer agent.

In another embodiment the Invention relates to methods for Increasingthe efficacy of an anti-cancer therapy given to a patient comprisingadministering an siNA of the Invention in combination with the therapy.Sais increase in efficacy may be up to or more than 10%, 20%, 30%, 40%,50%, 60%, 70%, 80% or 90% when compared to the efficacy of eitheradministration of siNA or the anti-cancer agent alone.

In a preferred embodiment, said siNA targets SEQ ID NO: 58 or SEQ ID NO:61 (for example, the siNA comprises a sense strand comprising a sequenceselected from SEQ ID NO: 162 or 165 respectively) or a variant thereof.In a further alternative embodiment, said siNA comprises a sense strand,wherein the sequence of the sense strand is SEQ ID NO 387 or a variantthereof, and an antisense strand, wherein the sequence of the antisensestrand is SEQ ID NO: 388 or a variant thereof. Alternatively, said siNAcomprises a sense strand, wherein the sequence of the sense strand isSEQ ID NO 393 or a variant thereof, and an antisense strand, wherein thesequence of the antisense strand is SEQ ID NO: 394 or a variant thereof.

In an alternative embodiment, said siNA targets SEQ ID NO: 267 or 278 ora variant thereof (for example, the siNA comprises a sense strandcomprising a sequence selected from SEQ ID NO: 356 or 367). In a furtheralternative embodiment, said siNA comprises a sense strand, wherein thesequence of the sense strand is SEQ ID NO 399 or a variant thereof, andan antisense strand, wherein the sequence of the antisense strand is SEQID NO: 400 or a variant thereof. Alternatively, said siNA comprises asense strand, wherein the sequence of the sense strand is SEQ ID NO 405or a variant thereof, and an antisense strand, wherein the sequence ofthe antisense strand is SEQ ID NO: 406 or a variant thereof.

In one embodiment, the anti-cancer agent is administered prior to,concurrently, or after administration of the siNA.

By “control” is meant herein either a cell or a patient administered nosiNA or a cell administered a vehicle, as described herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of targeting the LAT1 geneand/or ASCT2 gene by knocking down or inhibiting its expression as anovel strategy for cancer therapy. In particular, and according to afirst aspect of the present invention, there is provided the use of asiRNA for inhibiting LAT1 gene and/or ASCT2 gene expression in themanufacture of a medicament for treating or preventing cancer, whereinthe siRNA comprises a sense LAT1 or ASCT2 nucleic acid and an antisenseLAT1 or ASCT2 nucleic acid. The present invention also provides the useof a vector encoding the siRNA for inhibiting LAT1 and ASCT2 geneexpression in the manufacture of a medicament for treating or preventingcancer.

According to a second aspect of the present invention, there is provideda method of treating or preventing cancer comprising administering to anIndividual an effective amount of a siRNA that inhibits LAT1 and/orASCT2 gene expression, wherein the siRNA comprises a sense LAT1 or ASCT2nucleic acid and an antisense LAT1 or ASCT2 nucleic acid. The presentinvention also provides a method of treating or preventing cancercomprising administering to an individual an effective amount of avector encoding the siRNA that inhibits LAT1 and ASCT2 gene expression.

Overexpression of the LAT1 transporter, an isoform of system LNa⁺-independent neutral amino acid transporter, is a highly prevalentobservation in various forms of cancer. The present invention is basedon the surprising discovery that small interfering RNAs (siRNAs)selective for LAT1 are effective for treating cancer. In particular,bladder, blood, brain, colon, head and neck, kidney, liver, lung, lymphnode, mammary gland, metastatic, muscle, ovary, pancreas, prostate,skin, stomach and uterus cancer.

The siRNA or vector encoding the siRNA, or the medicament comprising thesiRNA or vector encoding the siRNA, may be administered to an individualby enteral administration (e.g., oral, rectal and intranasal),parenteral administration (e.g., intravascular administration, peri- andintra-tissue administration, subcutaneous injection or deposition,subcutaneous infusion intraocular administration and directadministration at or near the site of a tumour).

According to a third aspect of the present invention there is providedan in vitro method of inhibiting the expression of the LAT1 gene and/orASCT2 gene in a cell comprising contacting the cell with siNA thatinhibits LAT1 and/or ASCT2 gene expression as described herein. In oneembodiment, said siRNA comprises a sense LAT1 and/or ASCT2 nucleic acidand an anti-sense LAT1 and/or ASCT2 nucleic acid, wherein the sense LAT1or ASCT2 nucleic acid is substantially identical to a target sequencecontained within LAT1 or AST2 mRNA and the anti-sense LAT1 or ASCT2nucleic acid is complementary to the sense LAT1 or ASCT2 nucleic acid.The present invention also provides an in vitro method of inhibiting theexpression of the LAT1 and/or ASCT2 genes in a cell comprisingcontacting the cell with a vector encoding a siRNA that inhibits LAT1and/or ASCT2 gene expression, said siRNA comprises a sense LAT1 and/orASCT2 nucleic acid and an anti-sense LAT1 and/or ASCT2 nucleic acid,wherein the sense LAT1 or ASCT2 nucleic acid is substantially identicalto a target sequence contained within LAT1 or ASCT2 mRNA and theanti-sense LAT1 or ASCT2 nucleic acid is complementary to the sense LAT1or ASCT2 nucleic acid.

Expression of the gene may be inhibited by introduction of a doublestranded ribonucleic acid (dsRNA) molecule into the cell in an amountsufficient to inhibit expression of the LAT1 and/or ASCT2 genes.

The siRNAs used in the Invention are believed to cause the RNAi-mediateddegradation of LAT1 or ASCT2 mRNA so that the protein product of theLAT1 or ASCT2 gene is not produced or is produced in reduced amounts.The siRNAs used in the invention can be used to alter gene expression ina cell in which expression of LAT1 and/or ASCT2 is upregulated, e.g., asa result of malignant transformation of the cells. Binding of the siRNAto a LAT1 or ASCT mRNA transcript in a cell results in a reduction inLAT1 and ASCT2 production by the cell.

The term “siRNA” is used to mean a double stranded RNA molecule whichprevents translation of a target mRNA. Standard techniques ofintroducing siRNA into the cell are used, including those in which DNAis a template from which RNA is transcribed. The siRNA that inhibitsLAT1 or ASCT2 gene expression includes a sense LAT1 or ASCT2 nucleicacid sequence and an antisense LAT1 or ASCT2 nucleic acid sequence. ThesiRNA may be constructed such that a single transcript has both thesense and complementary antisense sequences from the target gene, e.g.,in the form of a hairpin.

The siRNA preferably comprises short double-stranded RNA that istargeted to the target mRNA, i.e., LAT1 mRNA or ASCT2 mRNA. The siRNAcomprises a sense RNA strand and a complementary antisense RNA strandannealed together by standard Watson-Crick base-pairing interactions(hereinafter “base-paired”). The sense strand comprises a nucleic acidsequence which is substantially identical to a target sequence containedwithin the LAT1 mRNA or ASCT2 mRNA.

The terms “sense/antisense sequences” and “sense/antisense strands” areused interchangeable herein to refer to the parts of the siRNA of thepresent invention that are substantially identical (sense) to the targetLAT1 and ASCT2 mRNA sequence or substantially complementary (antisense)to the target LAT1 and ASCT2 mRNA sequence.

As used herein, a nucleic acid sequence “substantially identical” to atarget sequence contained within the target mRNA is a nucleic acidsequence which is identical to the target sequence, or which differsfrom the target sequence by one or more nucleotides. Preferably, thesubstantially identical sequence is identical to the target sequence ordiffers from the target sequence by one, two or three nucleotides, morepreferably by one or two nucleotides and most preferably by only 1nucleotide. Sense strands which comprise nucleic acid sequencessubstantially identical to a target sequence are characterized in thatsiRNA comprising such a sense strand induces RNAi-mediated degradationof mRNA containing the target sequence. For example, an siRNA of theinvention can comprise a sense strand comprising a nucleic acid sequencewhich differs from a target sequence by one, two, three or morenucleotides, as long as RNAi-mediated degradation of the target mRNA isinduced by the siRNA.

The sense and antisense strands of the siRNA can comprise twocomplementary, single-stranded RNA molecules or can comprise a singlemolecule in which two complementary portions are base-paired and arecovalently linked by a single-stranded “hairpin” area. That is, thesense region and antisense region can be covalently connected via alinker molecule. The linker molecule can be a polynucleotide ornon-nucleotide linker. The siRNA can also contain alterations,substitutions or modifications of one or more ribonucleotide bases. Forexample, the present siRNA can be altered, substituted or modified tocontain one or more, preferably 0, 1, 2 or 3, deoxyribonucleotide bases.Preferably, the siRNA does not contain any deoxyribonucleotide bases.

The siRNA can comprise partially purified RNA, substantially pure RNA,synthetic RNA, or recombinantly produced RNA, as well as altered RNAthat differs from naturally-occurring RNA by the addition, deletion,substitution and/or alteration of one or more nucleotides. Suchalterations can include addition of non-nucleotide material, such as tothe end(s) of the siRNA or to one or more internal nucleotides of thesiRNA; modifications that make the siRNA resistant to nuclease digestion(e.g., the use of 2-substituted ribonucleotides or modifications to thesugar-phosphate backbone); or the substitution of one or more,preferably 0, 1, 2 or 3, nucleotides in the siRNA withdeoxyribonucleotides.

Degradation can be delayed or avoided by a wide variety of chemicalmodifications that include alterations in the nucleobases, sugars andthe phosphate ester backbone of the siRNAs. All of these chemicallymodified siRNAs are still able to induce siRNA-mediated gene silencingprovided that the modifications were absent in specific regions of thesiRNA and included to a limited extent. In general, backbonemodifications cause a small loss in binding affinity, but offer nucleaseresistance. Phosphorothioate (PS)- or boranophosphate (BS)-modifiedsiRNAs have substantial nuclease resistance. Silencing by siRNA duplexesis also compatible with some types of 2′-sugar modifications: 2′-H,2′-O-methyl, 2′-O-methoxyethyl, 2′-fluoro (2′-F), locked nucleic acid(LNA) and ethylene-bridge nucleic acid (ENA). Suitable chemicalmodifications are well known to those skilled in the art.

The siRNA used in the present invention is a double-stranded moleculecomprising a sense strand and an antisense strand, wherein the sensestrand comprises or consists of a ribonucleotide sequence correspondingto a LAT1 or ASCT2 target sequence, and wherein the antisense strandcomprises a ribonucleotide sequence which is complementary to said sensestrand, wherein said sense strand and said antisense strand hybridize toeach other to form said double-stranded molecule, and wherein saiddouble-stranded molecule, when introduced into a cell expressing theLAT1 and ASCT2 genes, inhibits expression of said genes. As indicatedfurther below, said LAT1 target sequence preferably comprises at leastabout 10 contiguous, more preferably 15 to 21, and most preferably about19 to 21 contiguous nucleotides selected from the group consisting offrom SEQ ID No 4, 6, 10, 13, 22, 34, 58, 61, 81, 83, 87, and 95 to 104.As indicated further below, said ASCT2 target sequence preferablycomprises at least about 10 contiguous, more preferably 15 to 21, andmost preferably about 19 to 21 contiguous nucleotides selected from thegroup consisting of from SEQ ID No 209, 216, 225, 226, 228, 235-238,245, 260, 264, 267, 271, 272, 278, 279, 281 to 297.

In one embodiment of the present invention, said sense strand andantisense strand of the siRNA molecule are covalently connected via alinker molecule. Said linker molecule may be a polynucleotide linker ora non-nucleotide linker. Preferably the linker is a loop sequence. Theloop sequence is preferably 3 to 23 nucleotide in length. Suitable loopsequences are described at http://www.ambion.com/techlib/tb/tb_506.htmland (Jacque et al., 2002). Preferred loop sequences include:

-   -   AUG: (Sui et al., 2002).    -   CCC, CCACC or CCACACC: (Paul et al., 2002).    -   UUCG: (Lee et al., 2002).    -   CTCGAG or AAGCUU: (Biology, 2003).    -   UUCAAGAGA: (Yu et al., 2002).

The loop sequence can be selected from group consisting of AUG, CCC,UUCG, CCACC. CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA. Preferably the loopsequence is UUCAAGAGA (“ttcaagaga” in DNA).

The siRNA used in the present invention can be obtained using a numberof techniques known to those of skill in the art. For example, the siRNAcan be chemically synthesized or recombinantly produced using methodsknown in the art, such as the Drosophila in vitro system described inU.S. published application 2002/0086356, the entire disclosure of whichis herein incorporated by reference. The siRNA may be chemicallysynthesized using appropriately protected ribonucleosidephosphoramidites and a conventional DNA/RNA synthesizer. The siRNA canbe synthesized as two separate, complementary RNA molecules, or as asingle RNA molecule with two complementary regions. Commercial suppliersof synthetic RNA molecules or synthesis reagents include Biospring(Frankfurt, Germany), Proligo (Hamburg, Germany), Dharmacon Research(Lafayette, Colo., USA), Thermo Fisher Scientific (Waltham, Mass. USA),Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) andSigma-Aidrich (St. Louis, Mo. USA).

The siRNA can also be expressed from recombinant circular or linear DNAvectors using any suitable promoter. Suitable promoters for expressingsiRNA from a vector include, for example, the U6 or H1 RNA pol IIIpromoter sequences and the cytomegalovirus promoter. Selection of othersuitable promoters is within the skill in the art. The vector can alsocomprise inducible or regulatable promoters for expression of the siRNAin a particular tissue or in a particular intracellular environment.

The siRNA expressed from a vector can either be isolated from culturedcell expression systems by standard techniques, or can be expressedintracellularty. The vector can be used to deliver the siRNA to cells invivo, e.g., by intracellularly expressing the siRNA in vivo. siRNA canbe expressed from a vector either as two separate, complementary RNAmolecules, or as a single RNA molecule with two complementary regions.Selection of vectors suitable for expressing the siRNA, methods forinserting nucleic acid sequences for expressing the siRNA into thevector, and methods of delivering the vector to the cells of interestare well known to those skilled in the art.

The siRNA can also be expressed from a vector intracelularty in vivo. Asused herein, the term “vector” means any nucleic acid- and/orviral-based technique used to deliver a desired nucleic acid. Any vectorcapable of accepting the coding sequences for the siRNA molecule(s) tobe expressed can be used, including plasmids, cosmids, naked DNA,optionally condensed with a condensing agent, and viral vectors.Suitable viral vectors include vectors derived from adenovirus (AV);adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV),Rhabdoviruses, murine leukemia virus); herpes virus, and the like. Thetropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Whenthe vector is a lentiviral vector it is preferably pseudotyped withsurface proteins from vesicular stomatitis virus, rabies virus, Ebolavirus or Mokola virus.

Vectors are produced for example by cloning a LAT1 or ASCT2 targetsequence into an expression vector so that operatively-linked regulatorysequences flank the LAT1 or ASCT2 sequence in a manner that allows forexpression (by transcription of the DNA molecule) of both strands (Leeet al., 2002). An RNA molecule that is antisense to LAT1 mRNA or ASCT2mRNA is transcribed by a first promoter (e.g., a promoter sequence 3′ ofthe cloned DNA) and an RNA molecule that is the sense strand for theLAT1 mRNA or the ASCT2 mRNA is transcribed by a second promoter (e. g.,a promoter sequence 5′ of the cloned DNA). The sense and antisensestrands hybridize in vivo to generate siRNA constructs for silencing ofthe LAT1 gene or ASCT2 gene. Alternatively, two vectors are utilized tocreate the sense and anti-sense strands of a siRNA construct. ClonedLAT1 or ASCT2 can encode a construct having secondary structure, e. g.,hairpins, wherein a single transcript has both the sense andcomplementary antisense sequences from the target gene. Such atranscript encoding a construct having secondary structure, willpreferably comprises a single-stranded ribonucleotide sequence (loopsequence) linking said sense strand and said antisense strand.

The siRNA is preferably isolated. As used herein, “isolated” meanssynthetic, or altered or removed from the natural state through humanintervention. For example, a siRNA naturally present in a IMng animal isnot “isolated,” but a synthetic siRNA, or a siRNA partially orcompletely separated from the coexisting materials of its natural stateis “Isolated.” An isolated siRNA can exist in substantially purifiedform, or can exist in a non-native environment such as, for example, acell into which the siRNA has been delivered. By way of example, siRNAwhich are produced inside a cell by natural processes, but which areproduced from an “isolated” precursor molecule, are themselves“isolated” molecules. Thus, an isolated dsRNA can be introduced into atarget cell, where it is processed by the Dicer protein (or itsequivalent) into isolated siRNA.

As used herein. “inhibit” means that the activity of the LAT1 or ASCT2gene expression product or level of the LAT1 or ASCT2 gene expressionproduct is reduced below that observed in the absence of the siRNAmolecule of the invention. The inhibition with a siRNA moleculepreferably is significantly below that level observed in the presence ofan Inactive or attenuated molecule that is unable to mediate an RNAiresponse. Inhibition of gene expression with the siRNA molecule ispreferably significantly greater in the presence of the siRNA moleculethan in its absence. Preferably, the siRNA inhibits the level of LAT1 orASCT2 gene expression by at least 10%, more preferably at least 50% andmost preferably at least 75%.

Preferably the siRNA molecule inhibits LAT1 or ASCT2 gene expression sothat growth of the cell containing the LAT1 or ASCT2 gene is inhibited.By inhibiting cell growth is meant that the treated cell proliferates ata lower rate or has decreased viability than an untreated cell. Cellgrowth is measured by proliferation assays known in the art.

As used herein, an “isolated nucleic acid” is a nucleic acid removedfrom its original environment (e. g., the natural environment ifnaturally occurring) and thus, synthetically altered from its naturalstate. In the present invention, isolated nucleic acid includes DNA,RNA, and derivatives thereof. When the isolated nucleic acid is RNA orderivatives thereof, base “t” should be replaced with “u” in thenucleotide sequences.

As used herein, the term “complementary” refers to Watson-Crick orHoogsteen base pairing between nucleotides units of a polynucleotide,and the term “binding” means the physical or chemical interactionbetween two polypeptides or compounds or associated polypeptides orcompounds or combinations thereof.

As used herein, the phrase “highly conserved sequence region” means anucleotide sequence of one or more regions in a target gene does notvary significantly from one generation to the other or from onebiological system to the other.

As used herein, the term “complementarity” or “complementary” means thata nucleic acid can form hydrogen bond(s) with another nucleic acidsequence by either traditional Watson-Crick or other non-traditionaltypes of interaction. In reference to the present invention, the bindingfree energy for a siRNA molecule with its complementary sequence issufficient to allow the relevant function of the nucleic acid toproceed, e.g., RNAi activity. For example, the degree of complementaritybetween the sense and antisense strand of the siRNA molecule can be thesame or different from the degree of complementarity between theantisense strand of the siRNA and the target RNA sequence.

A percent complementarity indicates the percentage of contiguousresidues in a nucleic acid molecule that can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence.Preferably the term “complementarity” or “complementary” means that atleast 90%, more preferably at least 95% and most preferably 100% ofresidues in a first nucleic acid sense can form hydrogen binds with asecond nucleic acid sequence.

Complementary nucleic acid sequences hybridize under appropriateconditions to form stable duplexes containing few (one or two) or nomismatches. Furthermore, the sense strand and antisense strand of thesiRNA can form a double stranded nucleotide or hairpin loop structure bythe hybridization. In a preferred embodiment, such duplexes contain nomore than 1 mismatch for every 10 matches. In an especially preferredembodiment, the sense and antisense strands of the duplex are fullycomplementary, i.e., the duplexes contain no mismatches.

As used herein, the term “cell” is defined using its usual biologicalsense. The cell can be present in an organism, e.g., mammals such ashumans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell canbe eukaryotic (e.g., a mammalian cell). The cell can be of somatic orgerm line origin, totipotent or pluripotent, dividing or non-dividing.The cell can also be derived from or can comprise a gamete or embryo, astem cell, or a fully differentiated cell. Preferably the cell is abladder, brain, colon, head and neck, kidney, liver, lung, lymph node,mammary gland, muscle, ovary, pancreas, skin or stomach cancer cell.

As used herein, the term “RNA” means a molecule comprising at least oneribonucleotide residue. By “ribonucleotide” is meant a nucleotide with ahydroxyl group at the 2′ position of a beta-D-ribo-furanose moiety. Theterm includes double stranded RNA, single stranded RNA, isolated RNAsuch as partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of the siRNAor internally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogues ofnaturally-occurring RNA. Preferably the term “RNA” consists ofribonucleotide residues only.

As used herein, the term“organism” refers to any living entity comprisedof at least one cell. A living organism can be as simple as, forexample, a single eukaryotic cell or as complex as a mammal, including ahuman being.

As used herein, the term “subject” means an organism, which is a donoror recipient of explanted cells or the cells themselves. “Subject” alsorefers to an organism to which the nucleic acid molecules of theinvention can be administered. The subject is preferably a mammal, e.g.,a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Mostpreferably the subject is a human.

As used herein, the term “biological sample” refers to any samplecontaining polynucleotides. The sample may be a tissue or cell sample,or a body fluid containing polynucleotides (e.g., blood, mucus,lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amnioticfluid, amniotic cord blood, urine, vaginal fluid and semen). The samplemay be a homogenate, lysate, extract, cell culture or tissue cultureprepared from a whole organism or a subset of its cells, tissues orcomponent parts, or a fraction or portion thereof. Lastly, the samplemay be a medium, such as a nutrient broth or gel in which an organism,or cells of an organism, have been propagated, wherein the samplecontains polynucleotides.

The invention relates to methods of inhibiting LAT1 and/or ACST2 geneexpression which causes the inhibition of cancer cell growth. Inparticular, the invention provides a method for inhibiting the growth ofa cancerous cell population comprising applying the LAT1 and/or ASCT2siRNA to said cancerous cell population. Cancer cell growth is inhibitedby contacting a cell with a composition of a LAT1 and/or ASCT2 siRNA.LAT1 and ASCT2 are amino acid transporter proteins that areoverexpressed in tumors such as bladder, brain, colon, head and neck,kidney, liver, lung, lymph node, mammary gland, muscle, ovary, pancreas,skin and stomach cancer. Growth of the cell expressing LAT1 or ASCT2 canbe inhibited by a LAT1 or ASCT2 siRNA. The cell may be further contactedwith a transfection-enhancing agent to enhance delivery of the siRNA orsiRNA encoding vector to the cell. Depending on the specific method ofthe present invention, the cell may be provided in vitro, in vivo or exvivo.

Sequence information regarding the human LAT1 gene (GenBank accessionNM_003486) was extracted from the NCBI Entrez nucleotide database. Up to104 mRNA segments were identified. Sequence information regarding thehuman ASCT2 gene (GenBank accession NM_001145144) was extracted from theNCBI Entrez nucleotide database. Up to 89 mRNA segments were identified.Methods for designing double stranded RNA having the ability to inhibitgene expression in a target cell are known. See for example, U.S. Pat.No. 6,506,559, and Elbashir et al., 2001, herein incorporated byreference in its entirety.

Selection of siRNA target sites can be performed as follows

-   -   1. Beginning with the ATG start codon of the transcript, scan        downstream for AA dinucleotide sequences. Record the occurrence        of each AA and the 3′ adjacent 19 nucleotides as potential siRNA        target sites. Tuschal et al. recommend against designing siRNA        to the 5′ and 3′ untranslated regions (UTRs) and regions near        the start codon (within 75 bases) as these may be richer in        regulatory protein binding sites. UTR-binding proteins and/or        translation initiation complexes may interfere with binding of        the siRNA endonuclease complex.    -   2. Compare the potential target sites to the appropriate genome        database (human, mouse, rat, etc.) and eliminate from        consideration any target sequences with significant homology to        other coding sequences. We suggest using BLAST, which can be        found on the NCBI server at :www.ncbi.nlm.nih.gov/BLAST/    -   3. Select qualifying target sequences (i.e., sequences having        over 55% GC content) for synthesis.

In one aspect of the invention, the length of the sense nucleic acid isat least 10 nucleotides and may be as long as the naturally-occurringLAT1 transcript. Preferably, the sense nucleic acid is less than 75, 50,or 25 nucleotides in length. It is further preferred that the sensenucleic acid comprises at least 19 nucleotides. Most preferably, thesense nucleic acid is 19-25 nucleotides in length. Examples of LAT1siRNA sense nucleic acids of the present invention which inhibit LAT1expression in mammalian cells include oligonucleotides comprising anyone of the following target sequences of the LAT1 gene: nucleotides145-165 (SEQ ID No 4), 217-237 (SEQ ID No 6), 466-486 (SEQ ID No 10),628-648 (SEQ ID No 13), 796-816 (SEQ ID No 22), 1243-1263 (SEQ ID No34), 525-545 (SEQ ID No 58), 624-644 (SEQ ID No 61), 1245-1265 (SEQ IDNo 81), 1316-1336 (SEQ ID No 83), 1410-1430 (SEQ ID No 87), 147-165 (SEQID No 95), 219-237 (SEQ ID No 96), 468-486 (SEQ ID No 97), 630-648 (SEQID No 98), 798-816 (SEQ ID No 99), 1247-1263 (SEQ ID No 100), 527-545(SEQ ID No 101), 1247-1265 (SEQ ID No 102), 1318-1336 (SEQ ID No 103),1412-1430 (SEQ ID No 104).

One hundred and four sequences, which set forth the sequence for onestrand of the double stranded is RNA, were generated for LAT-1. Theseincluded the following nucleotide sequences:

SEQ ID No 1 AAGCGGCGCGCGCTAGCGGCG SEQ ID No 2 AAGGAAGAGGCGCGGGAGAAGSEQ ID No 3 AAGAGGCGCGGGAGAAGATGC SEQ ID No 4 AAGATGCTGGCCGCCAAGAGCSEQ ID No 5 AAGAGCGCGGACGGCTCGGCG SEQ ID No 6 AACATCACGCTGCTCAACGGCSEQ ID No 7 AAGGAGGCAGGCTCGCCGGGG SEQ ID No 8 AAATCGGGCGGCGACTACGCCSEQ ID No 9 AATCGGGCGGCGACTACGCCT SEQ ID No 10 AAGCTCTGGATCGAGCTGCTCSEQ ID No 11 AAGCCGCTCTTCCCCACCTGC SEQ ID No 12 AAGCTCGTGGCCTGCCTCTGCSEQ ID No 13 AACTGCTACAGCGTGAAGGCC SEQ ID No 14 AAGGCCGCCACCCGGGTCCAGSEQ ID No 15 AAGCTCCTGGCCCTGGCCCTG SEQ ID No 16 AAGGGTGATGTGTCCAATCTASEQ ID No 17 AATCTAGATCCCAACTTCTCA SEQ ID No 18 AACTTCTCATTTGAAGGCACCSEQ ID No 19 AAGGCACCAAACTGGATGTGG SEQ ID No 20 AAACTGGATGTGGGGAACATTSEQ ID No 21 AACTGGATGTGGGGAACATTG SEQ ID No 22 AACATTGTGCTGGCATTATACSEQ ID No 23 AATTACTTGAATTTCGTCACA SEQ ID No 24 AATTTCGTCACAGAGGAAATGSEQ ID No 25 AAATGATCAACCCCTACAGAA SEQ ID No 26 AATGATCAACCCCTACAGAAASEQ ID No 27 AACCCCTACAGAAACCTGCCC SEQ ID No 28 AAACCTGCCCCTGGCCATCATSEQ ID No 29 AACCTGCCCCTGGCCATCATC SEQ ID No 30 AACCTGGCCTACTTCACCACCSEQ ID No 31 AACTATCACCTGGGCGTCATG SEQ ID No 32 AATGGGTCCCTGTTCACATCCSEQ ID No 33 AAGGCCACCTGCCCTCCATCC SEQ ID No 34 AAGGACATCTTCTCCGTCATCSEQ ID No 35 AACTTCTTCAGCTTCTTCAAC SEQ ID No 36 AACTGGCTCTGCGTGGCCCTGSEQ ID No 37 AAAGCCTGAGCTTGAGCGGCC SEQ ID No 38 AAGCCTGAGCTTGAGCGGCCCSEQ ID No 39 AAGGTGAACCTGGCCCTGCCT SEQ ID No 40 AACCTGGCCCTGCCTGTGTTCSEQ ID No 41 AAGACACCCGTGGAGTGTGGC SEQ ID No 42 AAAAACAAGCCCAAGTGGCTCSEQ ID No 43 AAACAAGCCCAAGTGGCTCCT SEQ ID No 44 AACAAGCCCAAGTGGCTCCTCSEQ ID No 45 AAGCCCAAGTGGCTCCTCCAG SEQ ID No 46 AAGTGGCTCCTCCAGGGCATCSEQ ID No 47 AAGCTCATGCAGGTGGTCCCC SEQ ID No 48 GGCGCCGGCGGCCGAGGAGAASEQ ID No 49 CCGGCGGCCGAGGAGAAGGAA SEQ ID No 50 GGAGAAGATGCTGGCCGCCAASEQ ID No 51 GAAGGAAGAGGCGCGGGAGAA SEQ ID No 52 GGAGAAGATGCTGGCCGCCAASEQ ID No 53 GGGCGTGACCCTGCAGCGGAA SEQ ID No 54 GACGCCCACGGGCGTGCTCAASEQ ID No 55 GCTCGGCACCACCATCTCCAA SEQ ID No 56 CTCGGCACCACCATCTCCAAASEQ ID No 57 CTCGCTGCCCGCCTTCCTCAA SEQ ID No 58 CTTCGCCACCTACCTGCTCAASEQ ID No 59 GGTGCCCGAGGAGGCAGCCAA SEQ ID No 60 GCTGCTGCTCACGGCCGTGAASEQ ID No 61 CGTGAACTGCTACAGCGTGAA SEQ ID No 62 GGATGCCTTTGCCGCCGCCAASEQ ID No 63 GGGCTTCGTCCAGATCGGGAA SEQ ID No 64 CGGGAAGGGTGATGTGTCCAASEQ ID No 65 TGTGTCCAATCTAGATCCCAA SEQ ID No 66 GATCCCAACTTCTCATTTGAASEQ ID No 67 CTTCTCATTTGAAGGCACCAA SEQ ID No 68 TTCTCATTTGAAGGCACCAAASEQ ID No 69 ACCAAACTGGATGTGGGGAA SEQ ID No 70 CTTTGCCTATGGAGGATGGAASEQ ID No 71 TGGAGGATGGAATTACTTGAA SEQ ID No 72 TGAATTTCGTCACAGAGGAAASEQ ID No 73 CGTCACAGAGGAAATGATCAA SEQ ID No 74 AAATGATCAACCCCTACAGAASEQ ID No 75 AATGATCAACCCCTACAGAAA SEQ ID No 76 GCTGGTGTACGTGCTGACCAASEQ ID No 77 CGTGGCCGTGGACTTCGGGAA SEQ ID No 78 GTCCTGCTTCGGCTCCGTCAASEQ ID No 79 TTCTTCGTGGGGTCCCGGGAA SEQ ID No 80 GCTGCTCTACGCCTTCTCCAASEQ ID No 81 GGACATCTTCTCCGTCATCAA SEQ ID No 82 CAACTTCTTCAGCTTCTTCAASEQ ID No 83 TGATCTGGCTGCGCCACAGAA SEQ ID No 84 GATCTGGCTGCGCCACAGAAASEQ ID No 85 TGAGCTTGAGCGGCCCATCAA SEQ ID No 86 TGAGCGGCCCATCAAGGTGAASEQ ID No 87 GATCGCCGTCTCCTTCTGGAA SEQ ID No 88 CTTCTTCGGGGTCTGGTGGAASEQ ID No 89 TTCTTCGGGGTCTGGTGGAAA SEQ ID No 90 TCTTCGGGGTCTGGTGGAAAASEQ ID No 91 CTTCGGGGTCTGGTGGAAAAA SEQ ID No 92 CGGGGTCTGGTGGAAAAACAASEQ ID No 93 CTGGTGGAAAAACAAGCCCAA SEQ ID No 94 CACGACCGTCCTGTGTCAGAASEQ ID No 95 AAGATGCTGGCCGCCAAGAGC SEQ ID No 96 AACATCACGCTGCTCAACGGCSEQ ID No 97 AAGCTCTGGATCGAGCTGCTC SEQ ID No 98 AACTGCTACAGCGTGAAGGCCSEQ ID No 99 AACATTGTGCTGGCATTATAC SEQ ID No 100 AAGGACATCTTCTCCGTCATCSEQ ID No 101 CTTCGCCACCTACCTGCTCAA SEQ ID No 102 GGACATCTTCTCCGTCATCAASEQ ID No 103 TGATCTGGCTGCGCCACAGAA SEQ ID No 104 GATCGCCGTCTCCTTCTGGAA

The LAT1 gene specificity was confirmed by searching NCBI BlastNdatabase. The siRNAs were chemically synthesized.

All of the forty-two purified siRNA duplexes were complexed withlipofectamine and added to the cells for 12 h in serum-free medium.Thereafter, cells were cultured for 72-96 h in serum-supplementedmedium, which was replaced by serum-free medium 24 h before theexperiments. A scrambled negative siRNA duplex was used as control.

The LAT1-siRNA is directed to a single target LAT1 gene sequence.Alternatively, the siRNA is directed to multiple target LAT1 genesequences. For example, the composition contains LAT1-siRNA directed totwo, three, four, five or more LAT1 target sequences. By LAT1 targetsequence is meant a nucleotide sequence that is identical to a portionof the LAT1 gene. The target sequence can include the 5′ untranslated(UT) region, the open reading frame (ORF) or the 3′ untranslated regionof the human LAT1 gene. Alternatively, the siRNA is a nucleic acidsequence complementary to an upstream or downstream modulator of LAT1gene expression. Examples of upstream and downstream modulators include,a transcription factor that binds the LAT1 gene promoter, a kinase orphosphatase that interacts with the LAT1 polypeptide, a LAT1 promoter orenhance.

LAT1-siRNA which hybridize to target mRNA decrease or inhibit productionof the LAT1 polypeptide product encoded by the LAT1 gene by associatingwith the normally single-stranded mRNA transcript, thereby interferingwith translation and thus, expression of the protein. Exemplary nucleicacid sequence for the production of LAT1-siRNA include the sequences ofnucleotides 145-165 (SEQ ID No 4), 217-237 (SEQ ID No 6), 466-486 (SEQID No 10), 628-648 (SEQ ID No 13), 796-816 (SEQ ID No 22), 1243-1263(SEQ ID No 34), 525-545 (SEQ ID No 58), 624-644 (SEQ ID No 61),1245-1265 (SEQ ID No 81), 1316-1336 (SEQ ID No 83), 1410-1430 (SEQ ID No87), 147-165 (SEQ ID No 95), 219-237 (SEQ ID No 96), 468-486 (SEQ ID No97), 630-648 (SEQ ID No 98), 798-816 (SEQ ID No 99), 1247-1263 (SEQ IDNo 100), 527-545 (SEQ ID No 101), 1247-1265 (SEQ ID No 102), 1318-1336(SEQ ID NP 103), 1412-1430 (SEQ ID No 104) as the target sequence. In afurther embodiment, in order to enhance the inhibition activity of thesiRNA, nucleotide “u” can be added to 3′ end of the antisense strand ofthe target sequence. Preferably at least 2, more preferably 2 to 10, andmost preferably 2 to 5 u's are added. The added u's form single strandat the 3′ end of the antisense strand of the siRNA.

The LAT1-siRNA can be directly introduced into the cells in a form thatis capable of binding to the mRNA transcripts. Alternatively, a vectorencoding the LAT1-siRNA can be introduced into the cells.

A loop sequence consisting of an arbitrary nucleotide sequence can belocated between the sense and antisense sequence in order to form ahairpin loop structure. Thus, the present invention also provides siRNAhaving the general formula 5′-[A]-[B]-[A]-3′, wherein [A] is aribonucleotide sequence corresponding to a target sequence of the LAT1gene. Preferably [A] is a sequence selected from the group consisting ofnucleotides 145-165 (SEQ ID No 4), 217-237 (SEQ ID No 6), 466-486 (SEQID No 10), 628-648 (SEQ ID No 13), 796-816 (SEQ ID No 22), 1243-1263(SEQ ID No 34), 525-545 (SEQ ID No 58), 624-644 (SEQ ID No 61),1245-1265 (SEQ ID No 81), 1316-1336 (SEQ ID No 83), 1410-1430 (SEQ ID No87), 147-165 (SEQ ID No 95), 219-237 (SEQ ID No 96), 468-486 (SEQ ID No97), 630-648 (SEQ ID No 98), 798-816 (SEQ ID No 99), 1247-1263 (SEQ IDNo 100), 527-545 (SEQ ID No 101), 1247-1265 (SEQ ID No 102), 1318-1336(SEQ ID No 103), 1412-1430 (SEQ ID No 104); [B] is a ribonucleotidesequence consisting of 3 to 23 nucleotides; and [A′] is a ribonucleotidesequence consisting of the complementary sequence of [A]. The region [A]hybridizes to [A′], and then a loop consisting of region [B] is formed.The loop sequence may be preferably 3 to 23 nucleotide in length.Suitable loop sequences are described athttp://www.ambion.com/techlib/tb/tb_506.html. Furthermore, loop sequenceconsisting of 23 nucleotides also provides active siRNA (Jacque et al.,2002). Preferred loop sequences included:

-   -   AUG: (Sui et al., 2002).    -   CCC, CCACC or CCACACC: (Paul et al., 2002).    -   UUCG: (Lee et al., 2002).    -   CTCGAG or AAGCUU: (Biology, 2003).    -   UUCAAGAGA: (Yu et al., 2002).

The loop sequence can be selected from group consisting of AUG, CCC,UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA. Preferably the loopsequence is UUCAAGAGA (“ttcaagaga” in DNA).

In a further aspect of the invention, the length of the sense nucleicacid is at least 10 nucleotides and may be as long as thenaturally-occurring ASCT2 transcript. Preferably, the sense nucleic acidis less than 75, 50, or 25 nucleotides in length. It is furtherpreferred that the sense nucleic acid comprises at least 19 nucleotides.Most preferably, the sense nucleic acid is 19-25 nucleotides in length.Examples of ASCT2 siRNA sense nucleic acids of the present inventionwhich inhibit ASCT2 expression in mammalian cells includeoligonucleotides comprising any one of the following target sequences ofthe ASCT2 gene: nucleotides 300-320 (SEQ ID No 209), 452-472 (SEQ ID No216), 773-793 (SEQ ID No 225), 776-796 (SEQ ID No 226), 830-850 (SEQ IDNo 228), 1122-1142 (SEQ ID No 235), 1123-1143 (SEQ ID No 236), 1124-1144(SEQ ID No 237), 1150-1170 (SEQ ID No 238), 769-789 (SEQ ID No 260),994-1014 (SEQ ID No 264), 1066-1086 (SEQ ID No 267), 1131-1151 (SEQ IDNo 271), 1154-1174 (SEQ ID No 272), 1264-1284 (SEQ ID No 278), 126-1288(SEQ ID No 279), 302-320 (SEQ ID No 281), 454-472 (SEQ ID NP 282),775-793 (SEQ ID No 283), 778-796 (SEQ ID No 284), 832-850 (SEQ ID No285), 1124-1142 (SEQ ID No 286), 1125-1143 (SEQ ID No 287), 1126-1144(SEQ ID No 288), 1152-1170 (SEQ ID No 289), 771-789 (SEQ ID No 291),996-1014 (SEQ ID No 292), 1068-1086 (SEQ ID No 293), 1133-1151 (SEQ IDNo 294), 1156-1174 (SEQ ID No 295), 1266-1284 (SEQ ID No 296), 1270-1288(SEQ ID No 297).

Eighty-nine sequences, which set forth the sequence for one strand ofthe double stranded is RNA, were generated for ASCT2. These included thefollowing nucleotide sequences:

SEQ ID No 209 AAGAGAGGAATATCACCGGAA SEQ ID No 210 AATATCACCGGAACCAGGGTGSEQ ID No 211 AACCAGGGTGAAGGTGCCCGT SEQ ID No 212 AAGGTGCCCGTGGGGCAGGAGSEQ ID No 213 AACATCCTGGGCTTGGTAGTG SEQ ID No 214 AAGCTGGGGCCTGAAGGGGAGSEQ ID No 215 AAGGGGAGCTGCTTATCCGCT SEQ ID No 216 AACTCCTTCAATGAGGCCACCSEQ ID No 217 AATGAGGCCACCATGGTTCTG SEQ ID No 218 AAGATCGTGGAGATGGAGGATSEQ ID No 219 AAGTACATTCTGTGCTGCCTG SEQ ID No 220 AAAAACCCCTACCGCTTCCTGSEQ ID No 221 AAAACCCCTACCGCTTCCTGT SEQ ID No 222 AAACCCCTACCGCTTCCTGTGSEQ ID No 223 AACCCCTACCGCTTCCTGTGG SEQ ID No 224 AAGTGCGTGGAGGAGAATAATSEQ ID No 225 AATAATGGCGTGGCCAAGCAC SEQ ID No 226 AATGGCGTGGCCAAGCACATCSEQ ID No 227 AAGCACATCAGCCGTTTCATC SEQ ID No 228 AACATGGACGGTGCCGCGCTCSEQ ID No 229 AAAGATCATCACCATCCTGGT SEQ ID No 230 AAGATCATCACCATCCTGGTCSEQ ID No 231 AAGCAGTCAACCTCCCGGTCG SEQ ID No 232 AACCTCCCGGTCGACCATATCSEQ ID No 233 AATGTAGAAGGTGACGCTCTG SEQ ID No 234 AAGGTGACGCTCTGGGGGCAGSEQ ID No 235 AAAATTACGTGGACCGTACGG SEQ ID No 236 AAATTACGTGGACCGTACGGASEQ ID No 237 AATTACGTGGACCGTACGGAG SEQ ID No 238 AAGCACAGAGCCTGAGTTGATSEQ ID No 239 AAGTGAAGAGTGAGCTGCCCC SEQ ID No 240 AAGAGTGAGCTGCCCCTGGATSEQ ID No 241 AAGGAAACCCCCTCCTCAAAC SEQ ID No 242 AAACCCCCTCCTCAAACACTASEQ ID No 243 AAACACTATCGGGGGCCCGCA SEQ ID No 244 AACACTATCGGGGGCCCGCAGSEQ ID No 245 AAGAGAGGAATATCACCGGAA SEQ ID No 246 TATCACCGGAACCAGGGTGAASEQ ID No 247 GCAGGAGGTGGAGGGGATGAA SEQ ID No 248 CTTTGGTGTGGCGCTGCGGAASEQ ID No 249 CTGCGGAAGCTGGGGCCTGAA SEQ ID No 250 GCTGCTTATCCGCTTCTTCAASEQ ID No 251 CCGCTTCTTCAACTCCTTCAA SEQ ID No 252 CATGTTCCTGGTGGCTGGCAASEQ ID No 253 ACTCTTTGCCCGCCTTGGCAA SEQ ID No 254 CTACTTCCTCTTCACCCGCAASEQ ID No 255 TACTTCCTCTTCACCCGCAAA SEQ ID No 256 CTTCCTCTTCACCCGCAAAAASEQ ID No 257 CACGCTGCCGCTGATGATGAA SEQ ID No 258 GATGAAGTGCGTGGAGGAGAASEQ ID No 259 GAAGTGCGTGGAGGAGAATAA SEQ ID No 260 GGAGAATAATGGCGTGGCCAASEQ ID No 261 GCCCATCGGCGCCACCGTCAA SEQ ID No 262 GCAGTCCTTGGACTTCGTAAASEQ ID No 263 AGCAGTCCTTGGACTTCGTAA SEQ ID No 264 CATCATCCTCGAAGCAGTCAASEQ ID No 265 ACTCTGGCCATCATCCTCGAA SEQ ID No 266 TGTACCGTCCTCAATGTAGAASEQ ID No 267 CCGGTCCTGTACCGTCCTCAA SEQ ID No 268 GGGGGCAGGACTCCTCCAAAASEQ ID No 269 TGGGGGCAGGACTCCTCCAAA SEQ ID No 270 CTGGGGGCAGGACTCCTCCAASEQ ID No 271 TGGACCGTACGGAGTCGAGAA SEQ ID No 272 ACAGAGCCTGAGTTGATACAASEQ ID No 273 GCCTGAGTTGATACAAGTGAA SEQ ID No 274 CTGCCAGTCCCCACTGAGGAASEQ ID No 275 CAGTCCCCACTGAGGAAGGAA SEQ ID No 276 GGAAGGAAACCCCCTCCTCAASEQ ID No 277 GAAGGAAACCCCCTCCTCAAA SEQ ID No 278 TGCCACGGTCGCCTCTGAGAASEQ ID No 279 ACGGTCGCCTCTGAGAAGGAA SEQ ID No 280 GAGAAGGAATCAGTCATGTAASEQ ID No 281 GAGAGGAATATCACCGGAA SEQ ID No 282 GCTGGGGCCTGAAGGGGAGSEQ ID No 283 TAATGGCGTGGCCAAGCAC SEQ ID No 284 TGGCGTGGCCAAGCACATCSEQ ID No 285 CATGGACGGTGCCGCGCTC SEQ ID No 286 AATTACGTGGACCGTACGGSEQ ID No 287 ATTACGTGGACCGTACGGA SEQ ID No 288 TTACGTGGACCGTACGGAGSEQ ID No 289 GCACAGAGCCTGAGTTGAT SEQ ID No 290 GAGAGGAATATCACCGGAASEQ ID No 291 AGAATAATGGCGTGGCCAA SEQ ID No 292 TCATCCTCGAAGCAGTCAASEQ ID No 293 GGTCCTGTACCGTCCTCAA SEQ ID No 294 GACCGTACGGAGTCGAGAASEQ ID No 295 AGAGCCTGAGTTGATACAA SEQ ID No 296 CCACGGTCGCCTCTGAGAASEQ ID No 297 GGTCGCCTCTGAGAAGGAA

The ASCT2 gene specificity was confirmed by searching NCBI BlastNdatabase. The siRNAs were chemically synthesized.

All of the forty-two purified siRNA duplexes were complexed withlipofectamine and added to the cells for 12 h in serum-free medium.Thereafter, cells were cultured for 72-96 h in serum-supplementedmedium, which was replaced by serum-free medium 24 h before theexperiments. A scrambled negative siRNA duplex was used as control.

The ASCT2-siRNA is directed to a single target ASCT2 gene sequence.Alternatively, the siRNA is directed to multiple target ASCT2 genesequences. For example, the composition contains ASCT2-siRNA directed totwo, three, four, five or more ASCT2 target sequences. By ASCT2 targetsequence is meant a nucleotide sequence that is identical to a portionof the ASCT2 gene. The target sequence can include the 5′ untranslated(UT) region, the open reading frame (ORF) or the 3′ untranslated regionof the human ASCT2 gene. Alternatively, the siRNA is a nucleic acidsequence complementary to an upstream or downstream modulator of ASCT2gene expression. Examples of upstream and downstream modulators include,a transcription factor that binds the ASCT2 gene promoter, a kinase orphosphatase that interacts with the ASCT2 polypeptide, a ASCT2 promoteror enhance.

ASCT2-siRNA which hybridize to target mRNA decrease or inhibitproduction of the ASCT2 polypeptide product encoded by the ASCT2 gene byassociating with the normally single-stranded mRNA transcript, therebyinterfering with translation and thus, expression of the protein.Exemplary nucleic acid sequence for the production of ASCT2-siRNAinclude the sequences of nucleotides 300-320 (SEQ ID No 209), 452-472(SEQ ID No 216), 773-793 (SEQ ID No 225), 776-796 (SEQ ID No 226),830-850 (SEQ ID No 228), 1122-1142 (SEQ ID No 235), 1123-1143 (SEQ ID No236), 1124-1144 (SEQ ID No 237), 1150-1170 (SEQ ID No 238), 769-789 (SEQID No 260), 994-1014 (SEQ ID No 264), 1066-1086 (SEQ ID No 267),1131-1151 (SEQ ID No 271), 1154-1174 (SEQ ID No 272), 1264-1284 (SEQ IDNo 278), 1268-1288 (SEQ ID No 279), 302-320 (SEQ ID No 281), 454-472(SEQ ID No 282), 775-793 (SEQ ID No 283), 778-796 (SEQ ID No 284),832-850 (SEQ ID No 285), 1124-1142 (SEQ ID No 286), 1125-1143 (SEQ ID No287), 1126-1144 (SEQ ID No 288), 1152-1170 (SEQ ID No 289), 771-789 (SEQID No 291), 996-1014 (SEQ ID No 292), 1068-1086 (SEQ ID No 293),1133-1151 (SEQ ID No 294), 1156-1174 (SEQ ID No 295), 1266-1284 (SEQ IDNo 296), 1270-1288 (SEQ ID No 297) as the target sequence. Furthermore,in order to enhance the inhibition activity of the siRNA, nucleotide “u”can be added to 3′ end of the antisense strand of the target sequence.Preferably at least 2, more preferably 2 to 10, and most preferably 2 to5 u's are added. The added u's form single strand at the 3′ end of theantisense strand of the siRNA.

The ASCT2-siRNA can be directly introduced into the cells in a form thatis capable of binding to the mRNA transcripts. Alternatively, a vectorencoding the ASCT2-siRNA can be introduced into the cells.

A loop sequence consisting of an arbitrary nucleotide sequence can belocated between the sense and antisense sequence in order to form ahairpin loop structure. Thus, the present invention also provides siRNAhaving the general formula 5′-[A]-[B]-[A′]-3′, wherein [A] is aribonucleotide sequence corresponding to a target sequence of the ASCT2gene. Preferably [A] is a sequence selected from the group consisting ofnucleotides 300-320 (SEQ ID No 209), 452-472 (SEQ ID No 216), 773-793(SEQ ID No 225), 776-796 (SEQ ID No 226), 830-850 (SEQ ID No 228),1122-1142 (SEQ ID No 235), 1123-1143 (SEQ ID No 236), 1124-1144 (SEQ IDNo 237), 1150-1170 (SEQ ID No 238), 769-789 (SEQ ID No 260), 994-1014(SEQ ID No 264), 1066-1086 (SEQ ID No 267), 1131-1151 (SEQ ID No 271),1154-1174 (SEQ ID No 272), 1264-1284 (SEQ ID No 278), 1268-1288 (SEQ IDNo 279), 302-320 (SEQ ID No 281), 454-472 (SEQ ID No 282), 775-793 (SEQID No 283), 778-796 (SEQ ID No 284), 832-850 (SEQ ID No 285), 1124-1142(SEQ ID No 286), 1125-1143 (SEQ ID No 287), 1126-1144 (SEQ ID No 288),1152-1170 (SEQ ID No 289), 771-789 (SEQ ID No 291), 996-1014 (SEQ ID No292), 1068-1086 (SEQ ID No 293), 1133-1151 (SEQ ID No 294), 1156-1174(SEQ ID No 295), 1266-1284 (SEQ ID No 296), 1270-1288 (SEQ ID No 297);[B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides; and[A] is a ribonucleotide sequence consisting of the complementarysequence of [A]. The region [A] hybridizes to [A], and then a loopconsisting of region [B] is formed. The loop sequence may be preferably3 to 23 nucleotide in length. Suitable loop sequences are described athttp://www.ambion.com/techlib/tbtb_506.html. Furthermore, loop sequenceconsisting of 23 nucleotides also provides active siRNA (Jacque et al.,2002). Preferred loop sequences included: AUG: (Sui et al., 2002).

-   -   CCC, CCACC or CCACACC: (Paul et al., 2002).    -   UUCG: (Lee et al., 2002).    -   CTCGAG or AAGCUU: (Biology, 2003).    -   UUCAAGAGA: (Yu et al., 2002).

The loop sequence can be selected from group consisting of AUG, CCC,UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA. Preferably the loopsequence is UUCAAGAGA (“ttcaagaga” in DNA).

The inventors have surprisingly found that siRNAs targeted to certaintarget sequences of the LAT1 gene or ASCT2 gene are particularlyeffective at inhibiting sodium-independent [¹⁴C]-L-leucine uptake orsodium-dependent [¹⁴C]-L-alanine uptake, respectively, LAT1 or ASCT2expression, cell growth and growth of tumors overexpressing LAT1 and/orASCT2 transporters.

In a specific embodiment of the present invention, the sense strand ofthe LAT1 siRNA used in the present invention comprises or consists of asequence selected from the group comprising SEQ ID No 6, No 22, No 34,No 58 and No 61. The siRNA also comprises a corresponding antisensestrand. The use of such an siRNA has been found to be particularlyeffective in inhibiting sodium-independent [¹⁴C]-L-leucine transport. Ina further embodiment, the sense strand of the LAT1 siRNA comprises orconsists of at least one sequence selected from the group comprising SEQID NO: 110, 126, 138, 162 and 165.

According to a another aspect of the present invention there is provideda siRNA comprising a sense LAT1 nucleic acid and an anti-sense LAT1nucleic acid, and the sense LAT1 nucleic acid is substantially identicalto a target sequence contained within LAT1 mRNA and the anti-sense LAT1nucleic acid is complementary to the sense LAT1 nucleic acid. The senseand antisense nucleic acids hybridize to each other to form adouble-stranded molecule.

The siRNA molecules of the present invention have the property toinhibit expression of the LAT1 gene when introduced into a cellexpressing said gene.

The siRNA molecules of the present invention have the property toinhibit cell growth when introduced into a cell expressing LAT1 gene.

The siRNA molecules of the present invention have the property toinhibit tumour growth when introduced into a tumour expressing LAT1gene.

In a specific embodiment of the present invention, the sense strand ofthe ASCT2 siRNA used in the present invention comprises or consists of asequence selected from the group comprising SEQ ID No 225, No 237, No267 and No 278. The siRNA also comprises a corresponding antisensestrand. The use of such an siRNA has been found to be particularlyeffective in inhibiting sodium-independent [¹⁴C]-L-leucine transport. Ina further embodiment, the sense strand of the LAT1 siRNA comprises orconsists of at least one sequence selected from the group comprising314, 326, 356 and 367.

According to a another aspect of the present invention there is provideda siRNA comprising a sense ASCT2 nucleic acid and an anti-sense ASCT2nucleic acid, and the sense ASCT2 nucleic acid is substantiallyidentical to a target sequence contained within ASCT2 mRNA and theanti-sense ASCT2 nucleic acid is complementary to the sense ASCT2nucleic acid. The sense and antisense nucleic acids hybridize to eachother to form a double-stranded molecule.

The siRNA molecules of the present invention have the property toinhibit expression of the ASCT2 gene when introduced into a cellexpressing said gene.

The siRNA molecules of the present invention have the property toinhibit cell growth when introduced into a cell expressing ASCT2 gene.

The siRNA molecules of the present invention have the property toinhibit tumour growth when introduced into a tumour expressing ASCT2gene.

When combined the siRNA-LAT1 and siRNA-ASCT2 of the present inventionhave the property to inhibit expression of the LAT1 and ASCT2 genes whenintroduced into a cell expressing said gene.

When combined the siRNA-LAT1 and siRNA-ASCT2 molecules of the presentinvention have the property to inhibit cell growth when introduced intoa cell expressing the LAT1 and ASCT2 genes.

When combined the siRNA-LAT1 and siRNA-ASCT2 molecules of the presentinvention have the property to inhibit tumour growth when introducedinto a tumour expressing and LAT1 ASCT2 genes.

Another aspect of the invention relates to nucleic acid sequences andvectors encoding the siRNA according to the fourth aspect of the presentinvention, as well as to compositions comprising them, useful, forexample, in the methods of the present invention. Compositions of thepresent invention may additionally comprise transfection enhancingagents. The nucleic acid sequence may be operably linked to an inducibleor regulatable promoter. Suitable vectors are discussed above.Preferably the vector is an adeno-associated viral vector.

The composition of the present invention may additionally comprise apharmaceutical agent for treating cancer, wherein the agent is differentfrom the siRNA. Preferably the pharmaceutical agent is selected from thegroup consisting of abarelix, amifostine, aminoglutethimide,anastrozole, bevacizumab, bicalutamide, bleomycin, bortezomib, busulfan,capecltabine, carboplatin, carmustine, cetuximab, chlorambucil,cispatlin, cladribine, cyclophosphamide, cytarabine, dacarbazine,dactinomycin, daunorubicln, docetaxel, doxorubicin, erlotinib,4′-epidoxorubicin, epirubicin, estramustine, etoposide, floxuridine,fludarabine, 5-fluorouracil, flutamide, gefitinib, gemcitabine,goserelin, hexamethylmelamine, hydroxyurea, Ifosfamide, imatinib,irinotecan, leuprolide, megestrol, melphalan, 6-mercatopurine,methotrexate, mitomycin, mitotane, mitoxantrone, oxaliplatin,paclitaxel, pentostatin, prednisone, procarbazine, rituximab,satraplatin, tamoxifen, temozolomide, teniposide, 6-thioguanine,thiotepa, topotecan, toremifen, trastuzumab, triptorelin, valrubicin,vinblastine, vincristine and vinolrebine.

Non-viral delivery siRNA systems involve the creation of nucleic acidtransfection reagents. Nucleic acid transfection reagents have two basicproperties. First, they must interact in some manner with the nucleicacid cargo. Most often this involves electrostatic forces, which allowthe formation of nucleic acid complexes. Formation of a complex ensuresthat the nucleic acid and transfedion reagents are presentedsimultaneously to the cell membrane. Complexes can be divided into threeclasses, based on the nature of the delivery reagent: lipoplexes;polyplexes; and lipopolyplexes. Lipoplexes are formed by the interactionof anionic nucleic acids with cationic lipids, polyplexes by interactionwith cationic polymers. Lipopolyplex reagents can combine the action ofcationic lipids and polymers to deliver nucleic acids. Addition ofhistone, poly-L-lysine and protamine to some formulations of cationiclipids results in levels of delivery that are higher than either lipidor polymer alone. The combined formulations might also be less toxic.The biocompatible systems most relevant to this purpose are non-viralbiodegradable nanocapsules designed especially according to the physicalchemistry of nucleic acids. They have an aqueous core surrounded by abiodegradable polymeric envelope, which provides protection andtransport of the siRNA into the cytosol and allow the siRNA to functionefficiently in vivo.

The present invention also provides a cell containing the siRNAaccording to the fourth aspect of the present invention or the vector ofthe present invention. Preferably the cell is a mammalian cell, morepreferably a human cell. It is further preferred that the cell is anisolated cell.

While the foregoing disclosure provides a general description of thesubject matter encompassed within the scope of the present invention,including methods, as well as the best mode thereof, of making and usingthis invention, the following examples are provided to further enablethose skilled in the art to practice this invention and to provide acomplete written description thereof. However, those skilled in the artwill appreciate that the specifics of these examples should not be readas limiting on the invention, the scope of which should be apprehendedfrom the claims and equivalents thereof appended to this disclosure.Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification, including reference tosequence database identifiers, are incorporated herein by reference intheir entirety. Unless otherwise specified, when reference to sequencedatabase identifiers is made, the version number is 1.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Unless context dictates otherwise, the descriptions and definitions ofthe features set out above are not limited to any particular aspect orembodiment of the invention and apply equally to all aspects andembodiments which are described. The invention is further described inthe following non-limiting examples.

The following examples further illustrate the present invention indetail but are not to be construed to limit the scope thereof.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Relative abundance of LAT1 protein and ASCT2 protein in humanliver carcinoma SK-HEP-1 cells, human bladder carcinoma T24 cells, humanfibrosarcoma HT-1080 cells and human colon cancer HTC-116 cells bywestern blot (relative to GAPDH).

FIG. 2. Relative abundance of LAT1 mRNA and ASCT2 mRNA in human livercarcinoma SK-HEP-1 cells, human bladder carcinoma T24 cells, humanfibrosarcoma HT-1080 cells and human colon cancer HTC-116 cells byRT-PCR (relative to GAPDH).

FIG. 3. Transport of ⁴C-L-leucine through LAT1 (sensitive to unlabelledL-leucine) and the transport of ¹⁴C-L-alanine through ASCT2 (sensitiveto unlabelled L-alanine). Significantly different from correspondingcontrol values (**** p<0.001).

FIG. 4. LAT1 mRNA relative abundance in liver carcinoma SK-HEP-1 cellstreated for 24 h with 10 nM siRNA-LAT1 (A) against nucleotide SEQs ID No6, 22, 34, 58 and 61 and (B) ASCT2 mRNA relative abundance in livercarcinoma SK-HEP-1 cells treated for 6 h with 10 nM siRNA-ASCT2 againstnucleotide SEQs ID No 225, 237, 267 and 278 on ASCT2 mRNA levels inhuman cancer cells (SK-HEP-1) using 0.5% Lipofectamine 2000 as thetransfecting agent at 24 h. Significantly different from correspondingcontrol values (* p<0.01) and values for SEQs ID No 34 (# p<0.05).

FIG. 5. [¹⁴C]-L-leucine (0.25 &M) (A and C) and [¹⁴C]-L-alanine (0.25μM) (B and D) uptake at initial rate of uptake (1 min) in livercarcinoma SK-HEP-1 cells treated for 6 h with 10 nM siRNA-LAT1 (A and C)against nucleotide SEQs ID No 6, 22, 34, 58 and 61 anti-LAT1 againstnucleotide SEQs ID No 6, 22, 34, 58 and 61 (A and C) and SEQs ID No 6,22, 34, 58 and 61 on and 10 nM siRNA-ASCT2 against nucleotide SEQs ID No225, 237, 267 and 278 using 0.4% Lipofectamine 2000 as the transfectingagent at 24 h (A and B) or 48 h (C and D). Significantly different fromcorresponding control values (* p<0.01), values for SEQs ID No 34 (#p<0.05) and values for SEQs ID No 267 (S p<0.05).

FIG. 6. 5′ DNA sense, 5′ sense siRNA and 3′ antisense siRNA-LAT1 againstnucleotide SEQs ID No 58, 58t, 58s1, 58s2, 61, 61t, 61s1 and 61s2.

FIG. 7. 5′ DNA sense, 5′ sense siRNA and 3′ antisense siRNA-ASCT2against nucleotide SEQs ID No 267, 267t, 267s1, 267s2, 278, 278t, 278s1and 278s2.

FIG. 8. [¹⁴C]-L-leucine (0.25 μM) (A) and [¹⁴C]-L-alanine (0.25 μM) (B)uptake at initial rate of uptake (1 min) in human fibrosarcoma HT-1080cells treated for 6 h with 5 nM anti-LAT1 against nucleotide SEQs ID No58, 58t, 61, 61t and and one commercially available siRNA (SI131011000from QIAGEN) 5 nM siRNA-ASCT2 against nucleotide SEQs ID No 267, 267t,278, 278t and one commercially available siRNA (S100097930, from QIAGEN)using 0.4% Lipofectamine 2000 as the transfecting agent at 48 h.Significantly different from corresponding control values (* p<0.01) andvalues for SEQ ID No 267t (* p<0.05).

FIG. 9. LAT1 mRNA (A and C) and ASCT2 mRNA (B and D) relative abundancein human colon cancer HTC-116 cells treated for 6 h with 5 or 25 nMsiRNA-LAT1 against nucleotide SEQs ID No 58, 61, 58t and 61t and ASCT2mRNA relative abundance in human colon cancer HTC-116 cells treated for6 h with 5 or 25 nM siRNA-ASCT2 against nucleotide SEQs ID No 267, 278,267t and 2781t using 0.5% Lipofectamine 2000 as the transfecting agentat 24 h (A and B) or 72 h (C and D). Significantly different fromcorresponding control values (* p<0.01).

FIG. 10. LAT1 mRNA relative abundance in human colon cancer HTC-116cells treated for 6 h with (A) 5, 25 and 45 nM siRNA-LAT1 againstnucleotide SEQs ID No 58t, using (B) 0.5% Lipofectamine 2000®,Lipofectamine RNAiMAX® or Injectin® as the transfecting agents at 72 h.Significantly different from corresponding control values (** p<0.01,*** p<0.02; *** p<0.001) and corresponding values with Injectin® (#p<0.05).

FIG. 11. Effect of transfecting agents Lipofectamine 2000® or Injectin®upon human colon cancer HTC-116 cell density after 72 h exposure.Significantly different from corresponding control values (* p<0.01,***p<0.02).

FIG. 12. siRNA complexation analysis by agarose gel electrophoresis inthe siRNA:Injectin® complexes at (A) 20 μL and (B) 80 μL complexvolumes. In panel A, electrophoresis of siRNA:Injectin® complexesrevealed that siRNA is fully complexed when the siRNA:Injectin® ratio is1:1 and the percentage of Injectin@ in the complex mixture is higherthan 0.9%. In panel B, siRNA complexation is not effective when thesiRNA:Injectin® ratio 1:1 is maintained but the amount of Injectin® inthe complex mixture is equal or lower than 0.2%.

FIG. 13. Effects of negative control siRNA commercial (from QIAGEN)sequences NC-S103650318 and NC-SI03650325 upon proliferation human coloncancer HTC-116 cells at 72 h exposure times using Injectin® (0.075 and0.1%) or Lipofedamine 2000® (0.25%) as the transfeding agent.

FIG. 14. Effects of (A) siRNA-LAT1 against nucleotide SEQs ID No 6, 22,34, 58, 58t, 58s1, 58s2, 61, 61t, 61s1, 61s2 and three commerciallyavailable siRNAs (SI131011000, HSS112005 and SASI_Hs01_00103513respectively from QIAGEN, Thermofisher Scientific and Sigma-Aldrich) and(B) siRNA-ASCT2 against nucleotide SEQs ID No 225, 237, 267, 267t,267s1, 267s2, 278, 278t, 278s1, 278s2 and one commercially availablesiRNA (SI100097930, from QIAGEN), respectively, upon proliferation ofhuman colon cancer HTC-116 cells at 72 h exposure times using 0.1%Injectin® as the transfecting agent. In panel A, values weresignificantly different from corresponding control values (* p<0.01),values for SEQ ID No 34 (#p<0.05) and values for SEQ ID No 58 ($p<0.05). In panel B, values were significantly different fromcorresponding control values (* p<0.01), values for SEQ ID No 267 (#p<0.05) and values for SEQ ID No 278 (S p<0.05).

FIG. 15. Effects of siRNA-LAT1 against nucleotide SEQs ID No 58t andsiRNA-ASCT2 against nucleotide SEQs ID No 267t and 278t uponproliferation human colon cancer HTC-116 cells at 72 h exposure timesusing increasing concentrations (0.037% to 0.15%) of Injectin® as thetransfecting agent. Significantly different from corresponding controlvalues (* p<0.05, ** p<0.01, *** p<0.02; **** p<0.001) and correspondingvalues with Injectin® (# p<0.05).

FIG. 16. Effects of 5-fluoruracil (5-FU; 3 and 10 μM), cisplatin (Cisp;3 and 10 μM) and oxaliplatin (Oxa; 1 and 3 μM) alone or in combinationwith siRNA-LAT1 against nucleotide SEQ ID No 58t (5 and 25 nM) uponproliferation of human colon cancer HTC-116 cells at 72 h exposure timesusing Increasing concentrations using 0.25% Lipofectamine 2000 as thetransfecting agent. Significantly different from corresponding controlvalues (# p<0.05, ####p<0.001) and corresponding values with theantineoplastic cytotoxic agent 5-FU, Cisp or Oxa (* p<0.05 ** p<0.01,*** p<0.02, **** p<0.001).

FIG. 17. Effects of 5-fluoruracil (5-FU; 10 μM) alone or in combinationwith anti-LAT1 SEQs ID No 581t and 61t (25 nM) upon proliferation ofhuman colon cancer HTC-116 cells at 72 h exposure times using 0.037%Injectin® as the transfecting agent. Significantly different fromcorresponding control values (#### p<0.001) and corresponding valueswith the antineoplastic cytotoxic agent 5-FU (** p<0.01, **** p<0.001).

FIG. 18. Effects of anti-LAT1 SEQs ID No 581t and 61t (25 nM) andanti-ASCT2 SEQ ID No 267t and 2781 alone or in combination uponproliferation of human colon cancer HTC-116 cells at 72 h exposure timesusing Injectin® as the transfecting agent. Significantly different fromcorresponding control values (* p<0.05 ** p<0.01, *** p<0.02, ****p<0.001).

FIG. 19. Effects of anti-LAT1 SEQs ID No 58, 58t, 58s1, 61, 61t, 61s1and two commercially available siRNAs (SI31011000 and HSS112005,respectively from QIAGEN and Thermofisher Scientific) upon (A) LAT1 mRNArelative abundance and (B) upon proliferation of human colon cancerHTC-116 cells at 72 h exposure times using 0.17% Lipofectamine 2000® or0.1% Injectin® as the transfecting agent. Significantly different fromcorresponding control values C p<0.01), values for SEQ ID No 58 (#p<0.05) and values for SEQ ID No 61 ($ p<0.05).

FIG. 20. Effects of anti-ASCT2 SEQs ID No 267, 267t, 267s1, 278, 278t,278s1 and one commercially available siRNA (S100097930, from QIAGEN)upon (A) ASCT2 mRNA relative abundance and (B) upon proliferation ofhuman colon cancer HTC-116 cells at 72 h exposure times using 0.17%Lipofectamine 2000® or 0.1% Injectin® as the transfecting agent.Significantly different from corresponding control values (* p<0.01),values for SEQ ID No 267 (#p<0.05) and values for SEQ ID No 2278 (*p<0.05).

FIG. 21. Representative western blot of LAT1 and ASCT2 proteins in thexenograft tumour model developed in immune deficient mice injectedsubcutaneously with human colon cancer HTC-116 cells.

FIG. 22. Relative abundance of LAT1 mRNA and ASCT2 mRNA by RT-PCR(relative to GAPDH) and relative abundance of LAT1 protein and ASCT2protein by western blot (relative to GAPDH) in the xenograft tumourmodel developed in immune deficient mice injected subcutaneously withhuman colon cancer HTC-116 cells.

FIG. 23. Relative tumour volume in the xenograft tumour model developedin immune deficient mice injected subcutaneously with human colon cancerHTC-116 cells and treated every other day with intratumoral injections(50 μL) of vehicle (Injectin®) or anti-LAT1 SEQ ID No 58t (10 μg) andanti-ASCT2 SEQ ID No 278t (10 μg). The reduction in relative tumorvolume induced by SEQ ID No 58t and SEQ ID No 278t was statisticallysignificant with p values of 0.0018 and 0.0051, respectively.

FIG. 24. Table of LAT1 preferred target sequences.

FIG. 25. Table of ASCT2 preferred target sequences.

FIG. 26. Table of LAT 1 target sequences and siRNA.

FIG. 27. Table of ASCT2 target sequences and siRNA.

MATERIALS AND METHODS

Cell Culture

SK-HEP-1, T24, HT-1080 and HCT-116 cell lines were maintained in ahumidified atmosphere of 5% CO₂ at 37° C. SK-HEP-1 cells were grown inRPMI-1640 (Sigma, St. Louis, Mo.) supplemented with 20% fetal bovineserum (FBS) (Gibco, UK), 100 U/mL penicillin G, 0.25 μg/mL amphotericinB, 100 μg/mL streptomycin (Gibco, UK), 25 mM sodium bicarbonate (Merck,Germany) and 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanosulfonic acid(HEPES) (Sigma, St. Louis, Mo.). T24 and HT-1080 cells were grown,respectively, in Dulbecco's Modified Eagle's Medium (DMEM)—high glucose(Sigma, St. Louis, Mo.) and DMEM—low glucose (Sigma, St. Louis, Mo.),supplemented with 10% FBS (Gibco, UK), 100 U/mL penicillin G, 0.25 μg/mLamphotericin B, 100 μg/mL streptomycin (Gibco, UK), 25 mM sodiumbicarbonate (Merck, Germany) and 25 mM HEPES (Sigma, St. Louis, Mo.).HCT-116 cells were grown in McCoy's 5A (Sigma, St. Louis, Mo.)supplemented with 10% FBS (Gibco, UK), 100 U/mL penicillin G, 0.25 μg/mLamphotericin B, 100 μg/mL streptomycin (Gibco, UK), 25 mM sodiumbicarbonate (Merck, Germany) and 25 mM HEPES (Sigma, St. Louis, Mo.).For all cell lines the medium was changed every 2 days, and cellsreached confluence 3-4 days after initial seeding. For subculturing,cells were dissociated with 0.25% trypsin-ethylenediaminetetraaceticacid (EDTA) (Sigma, St. Louis, Mo.), split 1:15 or 1:20 and subculturedin a 21-cm² growth area (Sarstedt, Germany).

LAT1 and ASCT2 Protein Expression

Cells were rinsed twice with cold phosphate-buffered saline (PBS) andincubated with 100 μL RIPA lysis buffer (154 mM NaCl, 65.2 mM TRIZMAbase, 1 mM EDTA, 1% NP-40 (IGEPAL), 6 mM sodium deoxycholate) containingprotease inhibitors: 1 mM PMSF, 1 μg/mL leupeptine and 1 μg/mLaprotinin; and phosphatase inhibitors: 1 mM Na₃VO₄ and 1 mM NaF. Cellswere scraped and briefly sonicated. Equal amounts of total protein (30μg) were separated on a 10% SDS-polyacrylamide gel andelectrotransferred to a nitrocellulose membrane in Tris-Glycine transferbuffer containing 20% methanol. The transblot sheets were blocked in 5%non-fat dry milk in Tris-buffered saline (TBS) for 60 min and thenincubated overnight, at 4° C., with the following antibodies: rabbitanti-LAT1 (1:1000; Cell Signalling); rabbit anti-ASCT2 (1:1000; CellSignalling); or mouse monoclonal anti-GAPDH (1:20,000; Santa CruzBiotechnology Inc.), diluted in 2.5% non-fat dry milk in TBS-Tween 20(0.1% vol/vol). The immunoblots were subsequently washed and incubatedwith fluorescently-labelled goat anti-rabbit (1:20,000; IRDye™ 800,Rockland) or fluorescently-labelled goat anti-mouse secondary antibody(1:20,000; AlexaFluor 680, Molecular Probes) for 60 min at roomtemperature (RT) and protected from light. Membranes were washed andimaged by scanning at both 700 nm and 800 nm with an Odyssey InfraredImaging System (LI-COR Biosciences).

LAT1 and ASCT2 Gene Expression

Total RNA was isolated and purified using the SV Total RNA IsolationSystem (Promega, USA) according to manufacturer's instructions. RNAquality and concentration were verified in the NanoDrop ND1000Spectrophotometer (Thermo Scientific, USA), and RNA integrity andgenomic DNA contamination were evaluated by agarose gel electrophoresis.Total RNA (1 μg) was converted into cDNA using the Maxima ScientificFirst Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific, USA),according to instructions. The following protocol was used: 1^(st) step,10 min at 25° C.; 2^(nd) step, 15 min at 50° C.; 3^(rd) step, 5 min at85° C. cDNA was used for qPCR analysis using Maxima SYBR Green qPCRMaster Mix (Thermo Scientific, USA) in the StepOnePlus instrument(Applied Biosystems, USA). QuantiTect Primer Assay for LAT1 and ASCT2and for the endogenous control gene GAPDH (Quiagen, Germany) were used.The qPCR reaction was performed in 96-well PCR plates (Sarstedt,Germany) as follows: one cycle of 10 min at 95° C., followed by 40 PCRcycles at 95° C. 15 s and 60° C. 60 s. A melting curve was madeimmediately after the qPCR, to demonstrate the specificity of theamplification. No template controls were always evaluated for eachtarget gene. Quantification cycle (Cq) values were generatedautomatically by the StepOnePlus 2.3 Software and the ratio of thetarget gene was expressed in comparison to the endogenous control geneGAPDH. Real-time PCR efficiencies were found to be between 90% and 110%.

LAT1 Activity

Cells were plated in 24-well plates (Sarstedt, Germany) and grown untilconfluence was reached. On the day of the experiment, cell culturemedium was aspirated and cells were preincubated for 15 min in Hanks'medium (NaCl 140 mM. KCl 5 mM, MgSO₄.7H₂O 0.8 mM, K₂HPO₄ 0.33 mM, KH₂PO₄0.44 mM, MgCl₂.6H₂O 1 mM, CaCl₂ 0.025 mM, Tris-HCl 9.75 mM, pH 7.4).Uptake was initiated by addition of Hanks' medium with 0.25 μM[¹⁴C]-L-leucine in the absence and in the presence of 3 mM unlabeledL-leucine. During preincubation and incubation cells were continuouslyshaken and maintained at 37° C. Uptake was terminated after 1 min byrapid removal of uptake solution by means of a vacuum pump connected toa Pasteur pipette, followed by a rapid wash with Hanks' medium.Subsequently, cells were solubilized in 0.1% vol/vol Triton X-100(dissolved in 5 mM Tris-HCl, pH 7.4), and radioactivity was measured byliquid scintillation counting.

ASCT2 Activity

Cells were plated in 24-well plates (Sarstedt, Germany) and grown untilconfluence was reached. On the day of the experiment, Cell culturemedium was aspirated and cells were preincubated for 15 min in Hanks'medium (ChCl 140 mM, KCl 5 mM, MgSO₄.7H₂O 0.8 mM, K₂HPO₄ 0.33 mM, KH₂PO₄0.44 mM, MgCl₂.6H₂O 1 mM, CaCl₂ 0.025 mM, Tris-HCl 9.75 mM, pH 7.4).Uptake was initiated by the addition of Hanks' medium with 0.25 μM[¹⁴C]-L-alanine in the absence and in the presence of 3 mM unlabeledL-alanin. During preincubation and incubation cells were continuouslyshaken and maintained at 37° C. Uptake was terminated after 1 min byrapid removal of uptake solution by means of a vacuum pump connected toa Pasteur pipette, followed by a rapid wash with Hanks' medium.Subsequently, cells were solubilized in 0.1% vol/vol Triton X-100(dissolved in 5 mM Tris-HCl, pH 7.4), and radioactivity was measured byliquid scintillation counting.

LAT1 Gene Silencing

Cells were plated in 24-well (Sarstedt, Germany) or 6-well plates(Sarstedt, Germany) or 96-well plates with black walls clear bottom (BDBiosciences, USA) and incubated 24 h under normal growth conditions.siRNAs against LAT1 and transfection agent were diluted at desiredconcentrations and mixed according to transfection agent manufacturer'sinstructions. The mixture was incubated 20 min at RT for siRNA-complexformation, after which it was added to the cells and incubated at 37°C., 5% CO₂. After the incubation period, serum and antibiotic wasrestored and cells were further incubated at normal conditions for thedesired time points until evaluation of LAT1 activity or LAT1 expression(immunoblotting and RT-qPCR).

ASCT2 Gene Silencing

Cells were plated in 24-well (Sarstedt. Germany) or 6-well plates(Sarstedt, Germany) or 96-well plates with black walls clear bottom (BDBiosciences, USA) and incubated 24 h under normal growth conditions.siRNAs against ASCT2 and transfection agent were diluted at desiredconcentrations and mixed according to transfection agent manufacturersinstructions. The mixture was incubated 20 min at RT for siRNA-complexformation, after which it was added to the cells and incubated at 37°C., 5% CO₂. After the incubation period, serum and antibiotic wasrestored and cells were further incubated at normal conditions for thedesired time points until evaluation of ASCT2 activity or ASCT2expression (immunoblotting and RT-qPCR).

Cell Proliferation Assay

Cell proliferation was measured using calcein-AM (Thermo FisherScientific, USA). The membrane permeant calcein-AM, a nonfluorescentdye, is taken up and converted by intracellular esterases to membraneimpermeant calcein, which emits green fluorescence. Cells were plated in96-well plates with black walls clear bottom (BD Biosciences, USA) andincubated 24 h under normal growth conditions. Cells were incubated withtest items at 37° C., 5% CO₂. After the incubation period, serum andantibiotic was restored and cells were further incubated at normalconditions during 72 h. After treatment with test substances or vehicle,cells were washed twice with Hanks' medium and loaded with 2 μMcalcein-AM in Hanks' medium, at at 37° C. for 30 min. Fluorescence wasmeasured at 485 nm excitation and 530 nm emission wavelengths in amicroplate spectrofluorometer (Gemini EM, Molecular Devices). Nineconsecutive fluorescence measurements are performed per well, to allowfluorescence readings in the whole area of the well, which was thenconsidered for the calculation of mean fluorescence per well. Todetermine minimum staining for calcein (calcein_(min)), eight wells weretreated with ethanol 30 min before calcein-AM addition. The percent cellnumber is calculated as [(calcein_(sample))/(calcein_(control))]×100.

Animals and Tumour Implantation

Human colon cancer HTC-116 cells grown in tissue culture and 10⁷ cellsper mouse were injected into the hind flank of female NMRI nu/nu mice.Once tumours have developed and tumour volumes reached randomisationcriteria, therapy will commence by every other day daily, intra-tumoralinjections. A vehicle treated group was included in the study ascontrol. Female immunodeficient NMRI nu/nu mice from Charles River wereused. The animals were delivered at the age of 4-6 weeks and are usedfor implantation after at least 1 week of quarantine. All animalsinterventions were performed in accordance with the European Directivenumber 86/609, and the rules of the “Guide for the Care and Use ofLaboratory Animals”, 7th edition, 1996, Institute for Laboratory AnimalResearch (ILAR), Washington. D.C. Only animals with unobjectionablehealth were selected to enter testing procedures. During theexperiments, animals were monitored at least daily. Each cage waslabelled with a record card indicating animal source, gender, and thedelivery date. Animals were numbered during tumour implantation or atthe initiation of a dose finding experiment.

The tumour volume was determined by a two-dimensional measurement withcallipers on the day of randomization (Day 0) and then twice weekly.Tumour volumes were calculated according to the following equation:

Tumour Vol[mm³ ]=a[mm]×b ²[mm²]×0.5

where “a” is the largest diameter and “b” is the perpendicular diameterof the tumour representing an idealized ellipsoid.

The relative volume of an Individual tumour on day X (RTV_(x)) wascalculated by dividing the absolute volume [mm³] of the respectivetumour on day X (T_(x)) by the absolute volume of the same tumour on theday of randomization, i. e. on day 0 (T₀), multiplied by 100, as shownby the following equation:

${{RTV}_{x}\lbrack\%\rbrack} = {\frac{T_{x}}{T_{0}} \times 100}$

RTVs were used for growth characterization and compound activity ratingas follows:

Rating RTV_(x) [%] CR Complete remission  ≦10 PR Partial remission >10;≦50 MR Minor remission >50; ≦75 NC No change  >75; ≦125 P Progression>125

Group median and range (alternatively geometric mean+/−SEM) of RTVs werecalculated, considering only the tumours of animals that were alive onthe day in question (for median). Group median (geometric mean) RTVswere used for drawing tumour growth curves and for treatment evaluation.

Tumour inhibition on a particular day (T/C_(x)) was calculated from themedian RTV of a test group and the median RTV of a control groupmultiplied by 100, as shown by the following equation:

$\frac{{T/{C_{x}\lbrack\%\rbrack}} = {{median}\mspace{14mu} {RTV}_{x}\mspace{14mu} {treated}\mspace{14mu} {group}}}{{median}\mspace{14mu} {RTV}_{x}\mspace{14mu} {control}\mspace{14mu} {group}} \times 100$

The optimum/minimum/best T/C [%] value recorded for a particular groupduring an experiment represents the maximum anti-tumour activity for therespective treatment and is rated as follows:

Rating T/C [%] − Inactive ≧65   +/− Borderline activity ≧50; ≦65 +Moderate activity ≧25; ≦50 ++ High activity ≧10; ≦25 +++ Very highactivity  ≧5; ≦10 ++++ Complete remission <5

Tumour volume doubling/quadrupling time (DT/QT) is defined as the timeinterval (in days) required for a group to reach a median RTV of200%/400% of the initial tumour volume. Growth delay is defined as thedifference in days between the tumour volume doubling and quadruplingtimes of a test group and the respective control group.

Non-Viral Delivery siRNA Systems

1. Liposomes carrying therapeutic siRNA-LAT1 agents are capable ofpassing through the membrane of the target cell to deliver cargo. Alarge number of lipids can be used for the synthesis of liposomes usedfor the delivery of siRNAs. Neutral lipids that can be complexed withsiRNA-LAT1 include DOPE(1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine), egg PC(phosphatidylchone), DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholineand DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamlne).

2. Cationic lipids that can be complexed with siRNA-LAT1 include DOTAP(1,2-dioley-3-trimetylammonium propane), CDAN(N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine)/DOPE, DC-Chol(3β-[N—(N′,N′-dimethylaminoethane)carbamoyl] cholesterol)/DOPE.DOTAP/DOPE, cationic lipid RPR209120(2-(3-[Bis-(3-amino-propyl)-amino]-propylamino)-N-ditetradecylcarbamoylme-thyl-acetamide).Galactosylated (Gal-C4-Chol/DOPE) liposomes/siRNA-LAT1 complex can alsoinduce gene silencing.

3. Cationic polymers can also be used in siRNA-LAT1 or siRNA-ASCT2delivery. These materials combine with anionic siRNA-LAT1 or siRNA-ASCT2to form a siRNA-LAT1-polymer complex or siRNA-ASCT2-polymer complex thatcan interact with the negatively charged cell surfaces through thecationic portion of the complex. Among the available polymers,polyethyleneimine (PEI) has the ability to bind strongly to negativelycharged siRNA-LAT1 or siRNA-ASCT2. Biodegradable polymers such aspoly(L-lysine) (PLL) are known for their lower toxicity and higherbiocompatibility than PEI. A derivative of PLL,poly[α-(4-aminobutyl)-L-glycolic acid] exhibits higher transfectionefficiency and lower immunogenicity and cytotoxicity than the originalPLL polymer and can be used with siRNA-LAT1 or siRNA-ASCT2.

4. Cationized gelatin microspheres can be prepared by chemicallycross-linking gelatin in the water-in-oil emulsion state. To impregnatesiRNA-LAT1 or siRNA-ASCT2 expression plasmid DNA into cationized gelatinmicrospheres, PBS containing siRNA-LAT1 expression plasmid DNA can bedropped onto freeze-dried cationized gelatin microspheres and then keptfor 24 h at 4° C.

5. Nanoparticles can be produced based on modified ionic gelation oftripolyphosphate (TPP) with chitosan. Two different types of chitosan(chitosan hydrochloride and glutamate) and each type with two differentmolecular weights can be used. Nanoparticles can be spontaneouslyobtained upon the addition of a TPP aqueous solution to chitosansolution under constant magnetic stirring at room temperature. Theparticles can then be incubated at room temperature for before use orfurther analysis. Nanoparticles are collected by centrifugation. Thesupernatants are discarded and nanoparticles are resuspended in filtereddistilled water. For the association of siRNA-LAT1 or siRNA-ASCT2 withthe chitosan-TPP nanoparticles (chitosan-TPP-siRNA-LAT1 orchitosan-TPP-siRNA-ASCT2), siRNA-LAT1 or siRNA-ASCT2 in double distilledwater is added to the TPP solution before adding this drop-wise to thechitosan solution under constant magnetic stirring at room temperature.The particles are then incubated at room temperature before use orfurther analysis.

6. Chitosan (114 kDa) was dissolved in sodium acetate buffer to obtain a0.2-1 mg/ml working solution range. Twenty microliters of siRNA-LAT1 orsiRNA-ASCT2 (20-250 μm range) was added to 1 ml of filtered chitosanwhile stirring and left for 1 h. To calculate specific N:P ratios(defined as the molar ratio of chitosan amino groups/RNA phosphategroups) a mass per phosphate of 325 Da was used for RNA and mass percharge of 167.88 for chitosan (84% deacetylation).

7. The siRNA-LAT1 or siRNA-ASCT2 can be encapsulated in stable nucleicacid lipid particles (SNALP) and administered by intravenous injection.The SNALP formulation contained the lipids 3-N-[(ω-methoxypoly(ethyleneglycol)₂₀₀₀)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-qminopropanone (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol.

8. The siRNA-LAT1 or siRNA-ASCT2 can be encapsulated in Injectin In VivoSiRNa Delivery Reagent (BioCelChallenge SAS, Toulon, France) andadministered by intravenous injection. The Injection formulationcontained the following mixture: 10 μg of siRNA in 10 pIL of glucosecontaining buffer, 40 μL with a sterile RNase-free water, 10 μL ofInjedin reagent. The mixture should be mix by pipetting up and down andincubated 15 minutes at room temperature before injection.

Example 1

LAT1 and ASCT2 Immunoblotting in Human Cancer Cells

The presence of LAT1 protein and ASCT2 was studied by means ofimmunoblotting using an antibodies raised against LAT1 and ASCT2. Asshown in FIG. 1, the antibody raised against LAT1 and ASCT2 recognizedthe presence of LAT1 and ASCT2 in all cancer cell lines.

Example 2

LAT1 and ASCT2 Gene Expression in Human Cancer Cells

The presence of LAT1 and ASCT2 mRNA was studied by means of Real-timePCR using primers against ASCT2. As shown in FIG. 2, LAT1 and ASCT2 geneexpression relative to the house keeping gene GADPH was found in allcancer cell lines.

Example 3

[¹⁴C]-L-Leucine and [¹⁴C]-L-Alanine Uptake

Sodium-independent [¹⁴C]-L-leucine (0.25 μM) uptake at initial rate ofuptake (1 min) in epithelial carcinoma cells was significantly (P<0.001)reduced by 3 mM unlabelled L-leucine as shown in FIGS. 3A-3D.Sodium-dependent [¹⁴C]-L-alanine (0.25 μM) uptake at initial rate ofuptake (1 min) in epithelial carcinoma cells was significantly (P<0.01)reduced by 3 mM unlabelled L-alanine, as shown in FIGS. 3E-3H.

Example 4

LAT1 and ASCT2 Gene Expression in Human Cancer Cells

As shown in FIG. 4A, treatment for 6 h of cells with the siRNA-LAT1against nucleotide sequences No 6, No 22, No 34, No 58 and No 61 (forexample, an siRNA comprising or consisting of SEQ ID NO: 110, 126, 138,162 and 165 respectively) decreased LAT1 mRNA relative abundance inliver carcinoma SK-HEP-1 cells at 24 h. The effects siRNA-LAT1 againstnucleotide SEQ No 58 were significantly greater (p<0.05) than those withSEQ No 34. As shown in FIG. 4B, treatment for 6 h of cells with thesiRNA-ASCT2 against nucleotide sequences No 225, No 237, No 267 and No278 (for example, an siRNA comprising or consisting of SEQ ID NO: 314,326, 356 and 367 decreased ASCT2 mRNA relative abundance in livercarcinoma SK-HEP-1 cells at 24 h. The effects siRNA-ASCT2 againstnucleotide SEQ No 278 were significantly greater (p<0.05) than thosewith SEQ No 267.

Example 5

[¹⁴C]-L-Leucine and [¹⁴C]-L-Alanine Uptake

As shown in FIGS. 5A and 5C, treatment for 6 h of cells with thesiRNA-LAT1 against nucleotide sequences No 6, No 22, No 34, No 58 and No61 (for example, an siRNA comprising or consisting of SEQ ID NO: 110,126, 138, 162 and 165 respectively) decreased [¹⁴C]-L-leucine (0.25 M)uptake at initial rate of uptake (1 min) in liver carcinoma SK-HEP-1cells at 24 h and 48 h. The effects siRNA-LAT1 against nucleotide SEQ No58 were significantly greater (p<0.05) than those with SEQ No 34. Asshown in FIGS. 5B and 5D, treatment for 6 h of cells with thesiRNA-ASCT2 against nucleotide sequences No 225, No 237, No 267 and No278 (for example, an siRNA comprising or consisting of SEQ ID NO: 314,326, 356 and 367 for 24 h [¹⁴C]-L-leucine (0.25 μM) uptake at initialrate of uptake (1 min) in liver carcinoma SK-HEP-1 cells at 24 h and 48h.

Example 6

Modifications of siRNA-LAT1 and siRNA-ASCT2

siRNA-LAT1 against nucleotide sequences No 58 and No 61 (for example, ansiRNA comprising or consisting of SEQ ID NO: 162 and 165 respectively)are shown in FIG. 6. (5′ DNA sense, 5′ sense siRNA and 3′ antisensesiRNA-LAT1 against nucleotide SEQs ID No 58, 58t, 58s1, 58s2, 61, 61t,61s1 and 61s2). siRNA-ASCT2 against nucleotide sequences No 267 and No278 (for example, an siRNA comprising or consisting of SEQ ID NO: 356and 367 respectively) are shown in FIG. 7. 5′ DNA sense, 5′ sense siRNAand 3′ antisense siRNA-ASCT2 against nucleotide SEQs ID No 267, 267t,267s1, 267s2, 278, 278t, 278s1 and 278s2

Example 7

[¹⁴C]-L-Leucine and [¹⁴C]-L-Alanine Uptake

As shown in FIG. 8A, treatment for 6 h of cells with the siRNA-LAT1against nucleotide sequences No 58, No 61, No 58t, No 61t as describedherein and the commercially available SI31011000 decreased[¹⁴C]-L-leucine (0.25 μM) uptake at initial rate of uptake (1 min) infibrosarcoma HT-1080 cells at 48 h. As shown in FIG. 8B, treatment for 6h of cells with the siRNA-ASCT2 against nucleotide sequences No 267, No278, No 267t and No 278t (for example, an siRNA comprising or consistingof SEQ ID NO: 356, 367 or an siRNA comprising or consisting of SEQ IDNO: 399 as the sense strand and SEQ ID NO: 400 as the antisense strandor SEQ ID NO: 405 as the sense strand and SEQ ID NO: 406 as theantisense strand respectively) and the commercially available S100097930decreased [¹⁴C]-L-alanine (0.25 μM) uptake at initial rate of uptake (1min) in fibrosarcoma HT-1080 cells at 48 h. The effects siRNA-ASCT2against nucleotide SEQ No 278t were significantly greater (p<0.05) thanthose with SEQ NO 267t.

Example 8

LAT1 and ASCT2 Gene Expression in Human Cancer Cells

As shown in FIGS. 9A and 9C, treatment for 6 h of cells with thesiRNA-LAT1 against nucleotide sequences No 58, No 61, No 58t and No 61t(for example, an siRNA comprising or consisting of SEQ ID NO: 162, 165,or an siRNA comprising or consisting of SEQ ID NO: 387 as the sensestrand and SEQ ID NO: 388 as the antisense strand or SEQ ID NO: 393 asthe sense strand and SEQ ID NO: 394 as the antisense strandrespectively) decreased LAT1 mRNA relative abundance in human coloncancer HTC-116 cells at 24 h or 48 h. As shown in FIGS. 9B and 9C,treatment for 6 h of cells with the siRNA-ASCT2 against nucleotidesequences No 267, No 278, No 267t and No 278t (for example, an siRNAcomprising or consisting of SEQ ID NO: 356, 367, or an siRNA comprisingor consisting of SEQ ID NO: 399 as the sense strand and SEQ ID NO: 400as the antisense strand or SEQ ID NO: 405 as the sense strand and SEQ IDNO: 406 as the antisense strand respectively) decreased ASCT2 mRNArelative abundance in human colon cancer HTC-116 cells at 24 h or 48 h.

Example 9

LAT1 Gene Expression in Human Cancer Cells

As shown in FIGS. 10A and 10B, treatment for 6 h of cells with thesiRNA-LAT1 against nucleotide sequences 58t (for example, an siRNAcomprising or consisting of SEQ ID NO: 387 as the sense strand and SEQID NO: 388 as the antisense strand) decreased LAT1 mRNA relativeabundance in human colon cancer HTC-116 cells in a concentrationdependent manner and when using 0.5% Lipofectamine 2000®, LipofectamineRNAiMAX® or Injectin® as the transfecting agents at 72 h.

Example 10

Cell Proliferation of Human Colon Cancer HTC-116 Cells, TransfectionAgents and Negative Controls

As shown in FIG. 11A, treatment for 6 h of human colon cancer HTC-116cells with 0.5% Lipofectamine 2000® for 72 h did not affect cellproliferation. By contrast, treatment of human colon cancer HTC-116cells with Injectin® did affect cell proliferation at 72 h in astatistically significant manner at 0.1%, 0.113 and 0.115%, as shown inFIG. 11B. Injectin is a lipid based siRNA transfection agent.Manufacture instructions recommend the use of 1 μg of siRNA per μL ofInjectin® reagent (ratio 1:1) where the volume of Injectin reagentcorresponds to 20% of the total complex mixture. For in vivo assays,siRNA:Injectin® complexes were prepared exactly as recommended by theproducer. For in vitro assays, the siRNA concentration used wasdownscaled and therefore the maintenance of 20% of Injectin® in thetotal siRNA:Injectin® complex mixture was unviable. For such a reason,siRNA complexation efficacy and siRNA:Injectin® transfection efficiencywere evaluated using different percentages of Injectin® in the complexmixture. Electrophoresis of siRNA:Injectin® complexes revealed thatsiRNA is fully complexed when the siRNA:Injectin® ratio is 1:1 and thepercentage of Injectin® in the complex mixture is higher than 0.9% (FIG.12A). However, siRNA complexation is not effective when thesiRNA:Injectin® ratio 1:1 is maintained but the amount of Injectin® Inthe complex mixture is equal or lower than 0.2% (FIG. 12B). This datasuggests that when the percentage of Injectin® in the complex is lowerthan 0.2% the recommended 1 μg of siRNA per μL of Injectin® ratio is nolonger effective. Therefore, the amount of Injectin® per μg of siRNAneeds to be increased in order to obtain a more efficientsiRNA:Injectin® complex. Similarly, an increase in the amount ofInjectin® in the complex mixture enhances the effect of siRNA sequencesupon cell proliferation (FIG. 15), as evidence of the enhancedtransfection efficiency of siRNA:Injectin® complexes with higher amountsof Injectin® in the siRNA transfection mixture. As shown in FIG. 13,negative control siRNA commercial (from QIAGEN) sequences NC-S103650318and NC-SI03650325 did not significantly affect the proliferation humancolon cancer HTC-116 cells at 72 h exposure times using Injectin® (0.075and 0.1%) or lipofectamine 2000 (0.25%) as the transfecting agent.

Example 11

Cell Proliferation of Human Colon Cancer HTC-116 Cells

As shown in FIG. 14A, treatment for 6 h of cells with the siRNA-LAT1against nucleotide sequence NO 6, No 22, NO 34, NO 58, NO 58t, NO 58s1,NO 58s2, NO 61, NO 61t, No 61s1, NO 61s2 as described herein, and threecommercially available siRNAs (SI131011000, HSS112005 andSASI_Hs01_00103513 respectively from QIAGEN, Thermofisher Scientific andSigma-Aidrich) decreased cell proliferation at 72 h exposure times using0.1% Injectin® as the transfecting agent. The effects siRNA-LAT1 againstnucleotide SEQ No 58 were significantly greater (p<0.05) than those withSEQ No 34 and the effects of siRNA-LAT1 against nucleotide SEQ No 58s1and 58s2 were significantly greater (p<0.05) than those with SEQ No 58.As shown in FIG. 14B, treatment for 6 h of cells with the siRNA-ASCT2against nucleotide sequence No 267, No 267t, No 267s1, No 267s2, No 278,No 278t, No 278s1, No 278s2 as described herein, and one commerciallyavailable siRNA (S100097930 from QIAGEN) decreased cell proliferation at72 h exposure times using 0.1% Injectin® as the transfecting agent. Theeffects siRNA-ASCT2 against nucleotide SEQ No 267t and No 267s2 weresignificantly greater (p<0.05) than those with SEQ No 267 and theeffects of siRNA-ASCT2 against nucleotide SEQ No 278s1 and No 278s2 weresignificantly greater (p<0.05) than those with SEQ No 278.

Treatment for 6 h of cells with the siRNA-LAT1 against nucleotidesequence No 58t (for example, an siRNA comprising or consisting of SEQID NO: 387 as the sense strand and SEQ ID NO: 388 as the antisensestrand), as shown in FIG. 15A, and siRNA-ASCT2 against nucleotidesequence No 267t and No 278t (for example, an siRNA comprising orconsisting of SEQ ID NO: 399 as the sense strand and SEQ ID NO: 400 asthe antisense strand or SEQ ID NO: 405 as the sense strand and SEQ IDNO: 406 as the antisense strand respectively), as shown in FIGS. 15B and18C respectively, decreased cell proliferation at 72 h exposure timesthat was greater the higher the concentration Injectin® in thesiRNA:Injectin® complexes. This was particularly evident with siRNA-LAT1against nucleotide sequence No 58t and siRNA-ASCT2 against nucleotidesequence No 278t.

Example 12

Cell Proliferation of Human Colon Cancer HTC-116 Cells in the Presenceof Cytotoxic Antineoplastic Agents

As shown in FIG. 16, the effects of the cytotoxic antineoplastic agents5-fluoruracil (5-FU; 3 and 10 μM), cisplatin (Cisp; 3 and 10 μM) andoxaliplatin (Oxa; 1 and 3 μM) alone upon proliferation of human coloncancer HTC-116 cells at 72 h exposure times were all significantlyenhanced in combination with the siRNA-LAT1 against nucleotide sequenceNo 58t, as described herein, in a concentration dependent manner. Asshown in FIG. 17A, the effects of the cytotoxic antineoplastic agents5-fluoruracil (5-FU; 10 μM) alone upon proliferation of human coloncancer HTC-116 cells at 72 h exposure times was enhanced by thesiRNA-LAT1 against nucleotide sequence No 58t as defined herein, but notby the siRNA-LAT1 against nucleotide sequence No 61t, as describedherein. Similarly, as shown in FIG. 17B, the enhancement of effects byhe cytotoxic antineoplastic agents 5-fluoruracil (5-FU; 10 μM) by thesiRNA-ASCT2 against nucleotide sequence No 278t, as described herein,was more marked than that by the siRNA-ASCT2 against nucleotide sequenceNo 267t, as described herein.

Example 13

Cell Proliferation of Human Colon Cancer HTC-116 Cells in the PresenceAnti-LA T1 and Anti-ASCT2 siRNAs

As shown in FIG. 18A, the significant decrease upon proliferation ofhuman colon cancer HTC-116 cells at 72 h exposure times by thesiRNA-LAT1 against nucleotide sequence No 58t, as described herein, wasenhanced by the siRNA-ASCT2 against nucleotide sequence No 278t, asdescribed herein, at non-efficacious conditions (0.037% Injectin®).Similarly, as shown in FIG. 18B, the significant effects uponproliferation of human colon cancer HTC-116 cells at 72 h exposure timesby the siRNA-LAT1 against nucleotide sequence No 58t, as describedherein, was enhanced by the siRNA-ASCT2 against nucleotide sequence No267t, as described herein, at non-efficacious conditions (0.037%Injectin®).

Example 14

LAT1 and ASCT2 Gene Expression and Cell Proliferation of Human ColonCancer HTC-116 Cells in the Presence Anti-LA T1 and Anti-ASCT2 siRNAs

As shown in FIG. 19A, treatment of cells with the siRNA-LAT1 againstnucleotide sequences NO 58, No 58t, No 58s1, No 61, No 61t, No 61s1 asdescribed herein, and two commercially available siRNAs (SI131011000 andHSS112005, respectively from QIAGEN and Thermofisher Scientific) for 6 hsignificantly decreased LAT1 mRNA relative abundance in human coloncancer HTC-116 cells though evidently with siRNA-LAT1 against nucleotidesequences No 58t and No 58s1. As shown in FIG. 19B, treatment of cellswith the siRNA-LAT1 against nucleotide sequences No 58, No 58t, No 58s1,No 61, No 61t, No 61s1, as described herein, and two commerciallyavailable siRNAs (SI31011000 and HSS112005, respectively from QIAGEN andThermofisher Scientific) for 6 h significantly decreased proliferationof human colon cancer HTC-116 cells at 72 h, though evidently withsiRNA-LAT1 against nucleotide sequences No 58, NO 58t and No 58s1.

As shown in FIG. 20A, treatment of cells with the siRNA-ASCT2 againstnucleotide sequences No 267, No 267t, No 2678s1, No 278, NP 278t. No278s1, as described herein, and one commercially available siRNAs(S100097930 from QIAGEN) for 6 h significantly decreased ASCT2 mRNArelative abundance in human colon cancer HTC-116 cells though evidentlywith siRNA-LAT1 against nucleotide sequences No 267t and No 278t. Asshown in FIG. 20B, treatment of cells with the siRNA-ASCT2 againstnucleotide sequences No 267, No 267t, No 2678s1, No 278, No 278t, No278s1, as described herein, and one commercially available siRNAs(S100097930 from QIAGEN) for 6 h significantly decreased proliferationof human colon cancer HTC-116 cells at 72 h, though evidently withsiRNA-ASCT2 against nucleotide sequence NO 278t.

Example 1

LAT1 and ASCT2 Immunoblotting in the Xenograft Tumour Model

The presence of LAT1 protein and ASCT2 was studied by means ofimmunoblotting using an antibodies raised against LAT1 and ASCT2. Asshown in FIG. 21, the antibody raised against LAT1 and ASCT2 recognizedthe presence of LAT1 and ASCT2 in both tumours (T1 and T2) derived fromhuman colon cancer HTC-116 cells.

Example 16

LAT1 and ASCT2 Gene Expression in Human Cancer Cells

The presence of LAT1 and ASCT2 mRNA was studied by means of Real-timePCR using primers against LAT1 and ASCT2. As shown in FIG. 22, LAT1 andASCT2 gene expression relative to the house keeping gene GADPH was foundin both tumours (T1 and T2) derived from human colon cancer HTC-116cells.

Example 17

Tumour Growth

As shown in FIG. 23, the relative tumour volume in the xenograft tumourmodel developed in immune deficient mice injected subcutaneously withhuman colon cancer HTC-116 cells and treated every other day withintratumoral injections (50 μL) of vehicle (Injectin®) or the siRNA-LAT1against nucleotide sequence No 58t (10 μg) and the siRNA-ASCT2 againstnucleotide sequence No 278t (10 μg), as described herein. The reductionin relative tumour volume by the siRNA-LAT1 against nucleotide sequenceNo 58t (10 μg) and the siRNA-ASCT2 against nucleotide sequence No 278twas statistically significant with p values of 0.0018 and 0.0051,respectively.

CONCLUSION

The treatment of cancer cells expressing LAT1 and/or ASCT2 transporterwith siRNA-LAT1 and/or siRNA-ASCT2 leads to a decrease in LAT1 and/orASCT2 protein and a decrease in [¹⁴C]-L-leucine uptake and[¹⁴C]-L-alanine uptake, which is accompanied by a decrease in cellproliferation. The decrease in cell viability and proliferation ofcancer cells induced by the siRNA-LAT1 and/or the siRNA-ASCT2 isaccompanied by apoptosis and a decrease in tumour growth and metastasispotential, as evidenced in nude mice subcutaneous tumours of human coloncancer HTC-116 cells.

Additional aspects of the invention will be apparent to those skilled inthe art, or may be learned from the practice of the invention. Theobjects and advantages of the invention may be realized and attained bymeans of the instrumentalities and combinations particularly pointed outin the appended claims.

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Boronophenylalanine, a boron delivery agent for boron neutron capturetherapy, is transported by ATB0,+, LAT1 and LAT2. Cancer Sci 106:279-286.

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1. An siNA (short interfering nucleic acid) molecule, wherein saidmolecule targets at least one sequence selected from SEQ ID NO: 1-SEQ IDNO: 104 and SEQ ID NO: 209-SEQ ID NO: 297 or a variant thereof or atleast one sequence complementary to a sequence selected from SEQ ID NO:1-SEQ ID NO: 104 and SEQ ID NO: 209-SEQ ID NO: 297 or a variant thereoffor use as a medicament.
 2. An siNA molecule according to claim 1,wherein the siNA specifically targets at least one sequence selectedfrom SEQ ID No 4, 6, 10, 13, 22, 34, 58, 61, 81, 83, 87 and 95 to 104 ora variant thereof, and wherein said molecule reduces expression of theLAT1 gene.
 3. An siNA molecule according to claim 1, wherein the siNAspecifically targets at least one sequence selected from SEQ ID NO: 209,216, 225, 226, 228, 235 to 238, 245, 260, 264, 267, 271, 272, 278, 279and 281 to 297 or a variant thereof, and wherein said molecule reducesexpression of the ASCT2 gene.
 4. An siNA molecule of claim 1, whereinsaid molecule is between 19 and 25 base pairs in length.
 5. An siNAmolecule of claim 1, wherein the siNA is selected from dsRNA, siRNA orshRNA.
 6. An siNA molecule of claim 5, wherein the siNA is siRNA.
 7. AnsiNA molecule of claim 1, wherein the siNA comprises 5′ and/or 3′overhangs.
 8. An siNA of claim 1, wherein the siNA comprises at leastone chemical modification.
 9. An siNA molecule of claim 1, wherein thesiNA molecule comprises a sense strand, and preferably an antisensestrand, wherein the sense strand comprises a sequence selected from SEQID NO: 105 to 208 and 298 to 386 or a variant thereof.
 10. An siNAmolecule of claim 9, wherein preferably the sense strand comprises atleast one sequence selected from 108, 110, 114, 117, 126, 138, 162, 165,185, 187, 191 and 199 to 208 or a variant thereof, and whereinpreferably, said molecule reduces expression of the LAT1 gene.
 11. AnsiNA molecule of claim 1, wherein preferable the sense strand comprisesat least one sequence selected from 298, 305, 314, 315, 317, 324-327,334, 349, 353, 356, 360, 361, 367, 368 and 370 to 386 or a variantthereof, and wherein preferably, said molecule reduces expression of theASCT 2 gene.
 12. An siNA molecule of claim 1, wherein the sense strandcomprises a sequence selected from SEQ ID NO: 387, 389, 391, 393, 395,397, 399, 401, 403, 405, 407 and 409 or a variant thereof and theantisense strand comprises a sequence selected from SEQ ID NO: 388, 390,392, 394, 306, 398, 400, 402, 404, 406, 408 and 410 respectively or avariant thereof.
 13. A pharmaceutical composition comprising at leastone siNA of claim 1 and a pharmaceutically acceptable carrier.
 14. Amethod for the treatment of cancer, the method comprising administeringthe siNA of claim
 1. 15. A method for the treatment of cancer, themethod comprising administering the pharmaceutical composition of claim13 to a patient in need thereof.
 16. The method of claim 14, wherein thecancer is selected from bladder, blood, brain, colon, head and neck,kidney, liver, lung, lymph node, mammary gland, metastatic, muscle,ovary, pancreas, prostate, skin, stomach and uterus cancer.
 17. Themethod of claim 15, wherein the cancer is selected from bladder, blood,brain, colon, head and neck, kidney, liver, lung, lymph node, mammarygland, metastatic, muscle, ovary, pancreas, prostate, skin, stomach anduterus cancer.
 18. A method of treating cancer comprising administratingto a patient in need thereof, the siNA of claim 1 in combination withone or more anti-cancer agents, preferably wherein the anti-cancer agentcomprises an anti-antineoplastic agent.
 19. A method of reducing cellproliferation, the method comprising contacting the cell with the siNAof claim
 1. 20. A nucleic acid construct or vector comprising a nucleicacid sequence encoding an siNA of claim 1.